Trehalose 6,6'-dimycolate promotes the survival of Mycobacterium tuberculosis in murine macrophages

The survival of Mycobacterium tuberculosis (MTB) in macrophages largely plays upon its ability to manipulate the host immune response to its benefit. Trehalose 6,6'-dimycolate (TDM) is a glycolipid found abundantly on the surface of MTB. Preliminary studies have shown that MTB lacking TDM have a lower survival rate compared to wild-type MTB in infection experiments, and that lysosomal colocalization with the phagosome occurs more readily in delipidated MTB infections. The purpose of this dissertation is to identify the possible mechanistic roles of TDM and its importance to the survival of MTB in macrophages. Our hypothesis is that TDM promotes the survival of MTB by targeting specific immune functions in host macrophages. Our first specific aim is to evaluate the effects of TDM on MTB in surface marker expression and antigen presentation in macrophages. We characterized the surface marker response in murine macrophages infected with either TDM-intact or TDM-removed MTB. We found that the presence of TDM on MTB inhibited the expression of surface markers which are important for antigen presentation and costimulation to T cells. Then we measured and compared the ability of macrophages infected by MTB with or without TDM to present Antigen 85B to hybridoma T cells. Macrophages infected with TDM-intact MTB were found to be less efficient at antigen presentation than TDM-removed MTB. Our second aim is to identify molecular mechanisms which may be targeted by TDM to promote MTB survival in macrophages. We measured macrophage responsiveness to IFN-γ before or after MTB infection and correlated SOCS production to the presence of TDM on MTB. Macrophages infected with TDM-intact MTB were found to be less responsive to IFN-γ. This may be attributed to the TDM-driven production of SOCS, which was found to affect phosphorylation of the JAK-STAT signaling pathway. We also identified the importance of TLR2 and TLR4 in the initiation of SOCS by TDM-intact MTB in host macrophages. In conclusion, our studies reveal new insights into how TDM regulates macrophages and their immune functions to aid in the survival of MTB.^

Differential expression of major histocompatibility complex class II genes on murine macrophages associated with T cell cytokine profile and protective/suppressive effects

Protective/suppressive major histocompatibility complex (MHC) class II alleles have been identified in humans and mice where they exert a disease-protective and immunosuppressive effect. Various modes of action have been proposed, among them differential expression of MHC class II genes in different types of antigen-presenting cells impacting on the T helper type 1 (Th1)–Th2 balance. To test this possibility, the expression of H-2 molecules from the four haplotypes H-2b, H-2d, H-2k, and H-2q was determined on bone marrow-derived macrophages (BMDMs) and splenic B cells. The I-Ab and I-Ek molecules, both well characterized as protective/suppressive, are expressed at a high level on almost all CD11b+ BMDMs for 5–8 days, after which expression slowly declines. In contrast, I-Ad, I-Ak, and I-Aq expression is lower, peaks over a shorter period, and declines more rapidly. No differential expression could be detected on B cells. In addition, the differential MHC class II expression found on macrophages skews the cytokine response of T cells as shown by an in vitro restimulation assay with BMDMs as antigen-presenting cells. The results indicate that macrophages of the protective/suppressive haplotypes express MHC class II molecules at a high level and exert Th1 bias, whereas low-level expression favors a Th2 response. We suggest that the extent of expression of the class II gene gates the back signal from T cells and in this way controls the activity of macrophages. This effect mediated by polymorphic nonexon segments of MHC class II genes may play a role in determining disease susceptibility in humans and mice.

Correction of murine galactosialidosis by bone marrow-derived macrophages overexpressing human protective protein/cathepsin A under control of the colony-stimulating factor-1 receptor promoter

Galactosialidosis (GS) is a human neurodegenerative disease caused by a deficiency of lysosomal protective protein/cathepsin A (PPCA). The GS mouse model resembles the severe human condition, resulting in nephropathy, ataxia, and premature death. To rescue the disease phenotype, GS mice were transplanted with bone marrow from transgenic mice overexpressing human PPCA specifically in monocytes/macrophages under the control of the colony stimulating factor-1 receptor promoter. Transgenic macrophages infiltrated and resided in all organs and expressed PPCA at high levels. Correction occurred in hematopoietic tissues and nonhematopoietic organs, including the central nervous system. PPCA-expressing perivascular and leptomeningeal macrophages were detected throughout the brain of recipient mice, although some neuronal cells, such as Purkinje cells, continued to show storage and died. GS mice crossed into the transgenic background reflected the outcome of bone marrow-transplanted mice, but the course of neuronal degeneration was delayed in this model. These studies present definite evidence that macrophages alone can provide a source of corrective enzyme for visceral organs and may be beneficial for neuronal correction if expression levels are sufficient.

Adapter proteins SLP-76 and BLNK both are expressed by murine macrophages and are linked to signaling via Fcγ receptors I and II/III

The SLP-76 (Src homology 2 domain-containing leukocyte protein of 76 kDa) adapter protein is expressed in T cells and myeloid cells, whereas its homologue BLNK (B cell linker protein) is expressed in B cells. SLP-76 and BLNK link immunoreceptor tyrosine-based activation motif-containing receptors to signaling molecules that include phospholipase C-γ, mitogen-activated protein kinases, and the GTPases Ras and Rho. SLP-76 plays a critical role in T cell receptor, FcɛRI and gpVI collagen receptor signaling, and participates in signaling via FcγR and killer cell inhibitory receptors. BLNK plays a critical role in B cell receptor signaling. We show that murine bone marrow-derived macrophages express both SLP-76 and BLNK. Selective ligation of FcγRI and FcγRII/III resulted in tyrosine phosphorylation of both SLP-76 and BLNK. SLP-76−/− bone marrow-derived macrophages display FcγR-mediated tyrosine phosphorylation of Syk, phospholipase C-γ2, and extracellular signal regulated kinases 1 and 2, and normal FcγR-dependent phagocytosis. These data suggest that both SLP-76 and BLNK are coupled to FcγR signaling in murine macrophages.

Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmania lipophosphoglycan in murine macrophages.

Lipophosphoglycan (LPG) glycoconjugates from promastigotes of Leishmania were not able to induce the expression of the cytokine-inducible nitric oxide synthase (iNOS) by the murine macrophage cell line, J774. However, they synergize with interferon gamma to stimulate the macrophages to express high levels of iNOS. This synergistic effect was critically time-dependent. Preincubation of J774 cells with the LPG glycans 4-18 h before stimulation with interferon gamma resulted in a significant reduction in the expression of iNOS mRNA and of NO synthesis, compared with cells preincubated with culture medium alone. The regulatory effect on the induction of iNOS by LPG is located in the LPG phosphoglycan disaccharide backbone. Synthetic fragments of this backbone had a similar regulatory effect on NO synthesis. Further, the production of NO by activated macrophages in the present system was correlated directly with the leishmanicidal capacity of the cells. These data therefore demonstrate that LPG glycoconjugates have a profound effect on the survival of Leishmania parasites through their ability to regulate the expression of iNOS by macrophages.

Receptor-mediated gene transfer into macrophages.

Gene transfer systems targeting various receptors have been developed to introduce functional genes into cells in culture and into intact animals. A synthetic molecular conjugate, consisting of mannosylated polylysine that exploits endocytosis via the macrophage mannose receptor, was constructed and complexed to expression plasmids containing either the Photinus pyralis luciferase or Escherichia coli beta-galactosidase (lacZ) reporter genes. The DNA complexes were used to transfect murine macrophages isolated from peritoneal exudates in vitro. Luciferase and beta-galactosidase activity was found in transfected cells in culture, whereas complexes consisting of an irrelevant plasmid bound to mannosylated polylysine or the expression plasmid bound to galactosylated polylysine resulted in no detectable transgene expression. Gene transfer was inhibited by the addition of excess mannosylated bovine serum albumin to the culture medium before transfection. Reporter genes were also transferred into macrophages residing in the spleen and liver of adult animals using this system. Luciferase activity was maximal at 4 days after transfection and decreased to lower levels by 16 days. Transgene expression conformed to the distribution of cells that had nonspecific esterase, a cytochemical marker for macrophages. Thus, this system can be used to introduce functional genes into macrophages and may be an approach to the treatment of storage diseases that affect the reticuloendothelial system.

Macrophage-colony-stimulating factor regulates expression of the integrins alpha 4 beta 1 and alpha 5 beta 1 by murine bone marrow macrophages.

We observed that when monocyte/macrophage precursors derived from murine bone marrow were treated with macrophage-colony-stimulating factor (M-CSF), there was a dose-dependent increase in both the number of adherent cells and the degree to which the cells were highly spread. Attachment was supported by fibronectin, but not by vitronectin or laminin, suggesting that the integrins alpha 4 beta 1 and/or alpha 5 beta 1 might mediate this event. Binding to fibronectin was blocked partially by antibodies to either integrin, and inhibition was almost complete when the antibodies were used in combination. By a combination of surface labeling with 125I and metabolic labeling with [35S]methionine and [35S]cysteine, we demonstrated that M-CSF treatment led to increased synthesis and surface expression of the two beta 1 integrins. Since attachment to fibronectin and/or stromal cells plays an important role in the maturation of other hematopoietic lineages, we propose that the action of M-CSF in the differentiation of immature monocytes/macrophages includes stimulated expression of the integrins alpha 4 beta 1 and alpha 5 beta 1, leading to interactions with components of the marrow microenvironment necessary for cell maturation.

The murine stk gene product, a transmembrane protein tyrosine kinase, is a receptor for macrophage-stimulating protein.

Macrophage-stimulating protein (MSP) was originally identified as an inducer of murine resident peritoneal macrophage responsiveness to chemoattractants. We recently showed that the product of RON, a protein tyrosine kinase cloned from a human keratinocyte library, is the receptor for MSP. Similarity of murine stk to RON led us to determine if the stk gene product is the murine receptor for MSP. Radiolabeled MSP could bind to NIH 3T3 cells transfected with murine stk cDNA (3T3/stk). Binding was saturable and was inhibited by unlabeled MSP but not by structurally related proteins, including hepatocyte growth factor and plasminogen. Specific binding to STK was demonstrated by cross-linking of 125I-labeled MSP to membrane proteins of 3T3/stk cells, which resulted in a protein complex with a molecular mass of 220 kDa. This radiolabeled complex comprised 125I-MSP and STK, since it could be immunoprecipitated by antibodies to the STK beta chain. Binding of MSP to stk cDNA-transfected cells induced tyrosine phosphorylation of the 150-kDa STK beta chain within 1 min and caused increased motile activity. These results establish the murine stk gene product as a specific transmembrane protein tyrosine kinase receptor for MSP. Inasmuch as the stk cDNA was cloned from a hematopoietic stem cell, our data suggest that in addition to macrophages and keratinocytes, a cell in the hematopoietic lineage may also be a target for MSP.

CpG DNA activates survival in murine macrophages through TLR9 and the phosphatidylinositol 3-kinase-Akt pathway

Bacterial CpG-containing (CpG) DNA promotes survival of murine macrophages and triggers production of proinflammatory mediators. The CpG DNA-induced inflammatory response is mediated via TLR9, whereas a recent study reported that activation of the Akt prosurvival pathway occurs via DNA-dependent protein kinase (DNA-PK) and independently. of TLR9. We show, in this study, that Akt activation and survival of murine bone marrow-derived macrophages (BMM) triggered by CpG-containing phosphodiester oligodeoxynucleotides or CpG-containing phosphorothioate oligodeoxynucleotides was completely dependent on TLR9. In addition, survival triggered by CpG-containing phosphodiester oligodeoxynucleotides was not compromised in BMM from SCID mice that express a catalytically inactive form of DNA-PK. CpG DNA-induced survival of BMM was inhibited by the PI3K inhibitor, LY294002, but not by the MEK1/2 inhibitor, PD98059. The effect of LY294002 was specific to survival, because treatment of BMM with LY294002 affected CpG DNA-induced TNF-alpha production only modestly. Therefore, CpG DNA activates macrophage survival via TLR9 and the PI3K-Akt pathway and independently of DNA-PK and MEK-ERK.

Damaging effects of advanced glycation end product in the murine macrophages cell line J774A.1.

The interaction of reducing sugars, such as aldose, with proteins and the subsequent molecular rearrangements, produces irreversible advanced glycation end-products (AGEs), a heterogeneous class of non-enzymatic glycated proteins or lipids. AGEs form cross-links, trap macromolecules and release reactive oxygen intermediates. AGEs are linked to aging, and increase in several related diseases. The aim of this study was to assess, in a murine macrophage cell line, J774A.1, the effects of 48 h of exposure to glycated serum containing a known amount of pentosidine, a well-known AGE found in the plasma and tissues of diabetic and uremic subjects. Fetal bovine serum was incubated with ribose (50 mm) for 7 days at 37 °C to obtain about 10 nmol/ml of pentosidine. The cytotoxic parameters studied were cell morphology and viability by neutral red uptake, lactate dehydrogenase release and tetrazolium salt test. In the medium and in the intracellular compartment, bound and free pentosidine were evaluated by HPLC, as sensitive and specific glycative markers, and thiobarbituric acid reactive substances (TBARs), as index of the extent of lipid peroxidation. Our results confirm that macrophages are able to take up pentosidine. It is conceivable that bound pentosidine is degraded and free pentosidine is released inside the cell and then into the medium. The AGE increase in the medium was combined with an increase in TBARs, meaning that an oxidative stress occurred; marked cytotoxic effects were observed, and were followed by the release of free pentosidine and TBARs into the culture medium.

IL-18 inhibits growth of murine orthotopic prostate carcinomas via both adaptive and innate immune mechanisms

Interleukin(IL)-18 is a pleiotrophic cytokine with functions in immune modulation, angiogenesis and bone metabolism. In this study, the potential of IL-18 as an immunotherapy for prostate cancer (PCa) was examined using the murine model of prostate carcinoma, RM1 and a bone metastatic variant RM1(BM)/B4H7-luc. RM1 and RM1(BM)/B4H7-luc cells were stably transfected to express bioactive IL-18. These cells were implanted into syngeneic immunocompetent mice, with or without an IL-18-neutralising antibody (αIL-18, SK113AE4). IL-18 significantly inhibited the growth of both subcutaneous and orthotopic RM1 tumors and the IL-18 neutralizing antibody abrogated the tumor growth-inhibition. In vivo neutralization of interferon-gamma (IFN-γ) completely eliminated the anti-tumor effects of IL-18 confirming an essential role of IFN-γ as a down-stream mediator of the anti-tumor activity of IL-18. Tumors from mice in which IL-18 and/or IFN-γ was neutralized contained significantly fewer CD4+ and CD8+ T cells than those with functional IL-18. The essential role of adaptive immunity was demonstrated as tumors grew more rapidly in RAG1−/− mice or in mice depleted of CD4+ and/or CD8+ cells than in normal mice. The tumors in RAG1−/− mice were also significantly smaller when IL-18 was present, indicating that innate immune mechanisms are involved. IL-18 also induced an increase in tumor infiltration of macrophages and neutrophils but not NK cells. In other experiments, direct injection of recombinant IL-18 into established tumors also inhibited tumor growth, which was associated with an increase in intratumoral macrophages, but not T cells. These results suggest that local IL-18 in the tumor environment can significantly potentiate anti-tumor immunity in the prostate and clearly demonstrate that this effect is mediated by innate and adaptive immune mechanisms.

Fracture healing via periosteal callus formation requires macrophages for both initiation and progression of early endochondral ossification

The distribution, phenotype, and requirement of macrophages for fracture-associated inflammation and/or early anabolic progression during endochondral callus formation were investigated. A murine femoral fracture model [internally fixed using a flexible plate (MouseFix)] was used to facilitate reproducible fracture reduction. IHC demonstrated that inflammatory macrophages (F4/80+Mac-2+) were localized with initiating chondrification centers and persisted within granulation tissue at the expanding soft callus front. They were also associated with key events during soft-to-hard callus transition. Resident macrophages (F4/80+Mac-2neg), including osteal macrophages, predominated in the maturing hard callus. Macrophage Fas-induced apoptosis transgenic mice were used to induce macrophage depletion in vivo in the femoral fracture model. Callus formation was completely abolished when macrophage depletion was initiated at the time of surgery and was significantly reduced when depletion was delayed to coincide with initiation of early anabolic phase. Treatment initiating 5 days after fracture with the pro-macrophage cytokine colony stimulating factor-1 significantly enhanced soft callus formation. The data support that inflammatory macrophages were required for initiation of fracture repair, whereas both inflammatory and resident macrophages promoted anabolic mechanisms during endochondral callus formation. Overall, macrophages make substantive and prolonged contributions to fracture healing and can be targeted as a therapeutic approach for enhancing repair mechanisms. Thus, macrophages represent a viable target for the development of pro-anabolic fracture treatments with a potentially broad therapeutic window...

Intracellular activities of Salmonella enterica in murine dendritic cells.

Dendritic cells (DC) efficiently phagocytose invading bacteria, but fail to kill intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium). We analysed the intracellular fate of Salmonella in murine bone marrow-derived DC (BM-DC). The intracellular proliferation and subcellular localization were investigated for wild-type S. Typhimurium and mutants deficient in Salmonella pathogenicity island 2 (SPI2), a complex virulence factor that is essential for systemic infections in the murine model and intracellular survival and replication in macrophages. Using a segregative plasmid to monitor intracellular cell division, we observed that, in BM-DC, S. Typhimurium represents a static, non-dividing population. In BM-DC, S. Typhimurium resides in a membrane-bound compartment that has acquired late endosomal markers. However, these bacteria respond to intracellular stimuli, because induction of SPI2 genes was observed. S. Typhimurium within DC are also able to translocate a virulence protein into their host cells. SPI2 function was not required for intracellular survival in DC, but we observed that the maturation of the Salmonella-containing vesicle is different in DC infected with wild-type bacteria and a strain deficient in SPI2. Our observations indicate that S. Typhimurium in DC are able to modify normal processes of their host cells.

Regulation of Chemokines, CCL3 and CCL4, by interferon gamma and Nitric oxide synthase 2 in mouse macrophages and during Salmonella enterica Serovar Typhimurium infection

Background. Interferon gamma (IFN-gamma) increases the expression of multiple genes and responses; however, the mechanisms by which IFN-gamma downmodulates cellular responses is not well understood. In this study, the repression of CCL3 and CCL4 by IFN-gamma and nitric oxide synthase 2 (NOS2) in macrophages and upon Salmonella typhimurium infection of mice was investigated. Methods. Small molecule regulators and adherent peritoneal exudates cells (A-PECs) from Nos2(-/-)mice were used to identify the contribution of signaling molecules during IFN-gamma-mediated in vitro regulation of CCL3, CCL4, and CXCL10. In addition, infection of bone marrow-derived macrophages (BMDMs) and mice (C57BL/6, Ifn-gamma(-/), and Nos2(-/-)) with S. typhimurium were used to gain an understanding of the in vivo regulation of these chemokines. Results. IFN-gamma repressed CCL3 and CCL4 in a signal transducer and activator of transcription 1 (STAT1)-NOS2-p38 mitogen activated protein kinase (p38MAPK)-activating transcription factor 3 (ATF3) dependent pathway in A-PECs. Also, during intracellular replication of S. typhimurium in BMDMs, IFN-gamma and NOS2 repressed CCL3 and CCL4 production. The physiological roles of these observations were revealed during oral infection of mice with S. typhimurium, wherein endogenous IFN-gamma and NOS2 enhanced serum amounts of tumor necrosis factor alpha and CXCL10 but repressed CCL3 and CCL4. Conclusions. This study sheds novel mechanistic insight on the regulation of CCL3 and CCL4 in mouse macrophages and during S. typhimurium oral infection.