miR-21a negatively modulates tumor suppressor genes PTEN and miR-200c and further promotes the transformation of M2 macrophages.

miR-21a is well-known to inhibit PTEN expression. We have previously shown that PTEN suppressed the transformation of M2 macrophages in the tumor microenvironment. Therefore, we hypothesized that miR-21a could influence M2 macrophage transformation by regulating PTEN expression. In this study, we identified how miR-21a reduced the expression of both PTEN mRNA and protein in murine macrophage cell lines and primary macrophages. Moreover, opposite effects were identified upon the use of a miR-21a inhibitor. Using a cytokine array, we identified the cytokines closely associated with miR-21a-mediated macrophage transformation to the M2 phenotype. miR-21a mimics could also enhance the migratory ability of murine breast cancer 4T1 cells, the growth of breast cancer in vivo and CD206 intratumor expression. In addition, quantitative PCR (qPCR) and methylation-specific PCR analysis showed that miR-21a enhanced miR-200c methylation and then decreased miR-200c and PTEN expression. These effects could be reversed by treatment with 5'-Aza, a DNA-demethylating agent. MiR-200c was predicted to target the PTEN 3'UTR, but qPCR illustrated the miR-200c mimic that increased PTEN expression, and 5'-Aza could enhance its effect. The above results indicate that miR-21a negatively modulates two tumor suppressor genes, miR-200c and PTEN, thereby promoting M2 macrophage transformation. This demonstrates that miR-21a represents a novel target for improving the overall tumor microenvironment.

Oxidized Low-Density Lipoprotein Loading of Macrophages Downregulates TLR-Induced Proinflammatory Responses in a Gene-Specific and Temporal Manner through Transcriptional Control.

Hypercholesterolemia is a key risk factor for atherosclerosis and leads to the uptake of native and oxidized low-density lipoprotein (oxLDL) by macrophages (Mϕs) and foam cell formation. Inflammatory processes accompany Mϕ foam cell formation in the artery wall, yet the relationship between Mϕ lipid loading and their response to inflammatory stimuli remains elusive. We investigated proinflammatory gene expression in thioglycollate-elicited peritoneal Mϕs, bone marrow-derived Mϕs and dendritic cells, and RAW264.7 cells. Loading with oxLDL did not induce peritoneal Mϕ apoptosis or modulate basal-level expression of proinflammatory genes. Upon stimulation of TLR4, the rapid induction of IFN-ß was inhibited in cells loaded with oxLDL, whereas the induction of other proinflammatory genes by TLR4 (LPS), TLR3 (polyriboinosinic-polyribocytidylic acid), TLR2 (Pam3CSK4), and TLR9 (CpG) remained comparable within the first 2 h. Subsequently, the expression of a subset of proinflammatory genes (e.g., IL-1ß, IL-6, CCL5) was reduced in oxLDL-loaded cells at the level of transcription. This phenomenon was partially dependent on NF erythroid 2-related factor 2 (NRF2) but not on nuclear liver X receptors α and ß (LXRα,ß), peroxisome proliferator-activated receptor-γ (PPARγ), and activating transcription factor 3 (ATF3). LPS-induced NF-κB reporter activity and intracellular signaling by NF-κB and MAPK pathways were comparable in oxLDL-loaded Mϕs, yet the binding of p65/RelA (the prototypic NF-κB family member) was reduced at IL-6 and CCL5 promoters. This study revealed that oxLDL loading of Mϕs negatively regulates transcription at late stages of TLR-induced proinflammatory gene expression and implicates epigenetic mechanisms such as histone deacetylase activity.

The multiple chemokine-binding bovine herpesvirus 1 glycoprotein G (BHV1gG) inhibits polymorphonuclear cell but not monocyte migration into inflammatory sites.

Chemokines facilitate the recruitment of inflammatory cells into tissues, contributing to target organ injury in a wide range of inflammatory and autoimmune diseases. Targeting either single chemokines or chemokine receptors alters the progression of disease in animal models of rheumatoid arthritis and lupus with varying degrees of efficacy but clinical trials in humans have been less successful. Given the redundancy of chemokine-chemokine receptor interactions, targeting of more than one chemokine may be required to inhibit active inflammatory disease. To test the effects of multiple-chemokine blockade in inflammation, we generated an adenovirus expressing bovine herpesvirus 1 glycoprotein G (BHV1gG), a viral chemokine antagonist that binds to a wide spectrum of murine and human chemokines, fused to the Fc portion of murine IgG2a. Administration of the adenovirus significantly inhibited thioglycollate-induced migration of polymorphonuclear leukocytes into the peritoneal cavity of BALB/c mice and reduced both clinical severity and articular damage in K/BxN serum transfer-induced arthritis. However, treatment with BHV1gG-Ig fusion protein did not prevent monocyte infiltration into the peritoneum in the thioglycollate model and did not prevent renal monocyte infiltration or nephritis in lupus-prone NZB/W mice. These observations suggest that the simultaneous inhibition of multiple chemokines by BHV1gG has the potential to interfere with acute inflammatory responses mediated by polymorphonuclear leukocytes, but is less effective in chronic inflammatory disease mediated by macrophages.

Thiomer-coated liposomes harbor permeation enhancing and efflux pump inhibitory properties.

An ideal oral drug carrier should facilitate drug delivery to the gastrointestinal tract and its absorption into the systemic circulation. To meet these requirements, we developed a thiomer-coated liposomal delivery system composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and a maleimide-functionalized lipid, to which chitosan-thioglycolic acid (CS-TGA) was covalently coupled. In addition to conventional 77 kDa CS-TGA (CS-TGA77), we tested the 150 kDa homologue (CS-TGA150) as well as an S-protected version of this polymer (CS-TGA150-MNA), in which some of the free SH-groups are conjugated with 6-mercaptonicotinamide to protect them from oxidation. Coupling of CS-TGA to the liposomal surface led to an increase in the particle size of at least 150 nm and an increase in the zeta potential from approximately -33 mV to a maximum of about +36 mV, depending on the polymer. As revealed by fluorescence dequenching the formulations have a storage stability of at least two weeks without releasing any encapsulated compounds. In simulated gastric fluid, the system was shown to be stable over 24 h, while in simulated intestinal fluid, a slow, sustained release of encapsulated compounds was observed. According to our experiments, thiomer-coated liposomes did not induce immunogenic reactions after an oral administration to mice. To evaluate the permeation enhancing and efflux pump inhibiting properties of CS-TGA coated liposomes we monitored the transport of fluoresceinisothiocyanate-dextran (FD(4)) and rhodamine-123 (Rho-123), respectively, through rat small intestine. Permeation studies showed a 2.8-fold higher permeation of FD(4) in the presence of CS-TGA77 coated liposomes and an even 4-fold higher permeation in the presence of CSA-TGA150-MNA coated liposomes. The latter also performed best when we evaluated P-glycoprotein inhibiting properties by monitoring the transport of Rho-123, revealing a 4.2-fold enhancement respective to the buffer control. Taken together, thiomer-coated liposomes were shown to protect encapsulated drugs in the stomach, slowly release them in the small intestine and enhance their absorption through the intestinal tissue by opening tight junctions and inhibiting efflux pumps.

JNK-1 deficiency limits macrophage-mediated antigen-induced arthritis.

OBJECTIVE: To elucidate the nonredundant roles of JNK-1 and JNK-2 in antigen-induced arthritis (AIA). METHODS: Mice that were genetically disrupted in Jnk1 or Jnk2 were primed by injection of methylated bovine serum albumin (mBSA) in Freund's complete adjuvant and then challenged on day 21 by intraarticular injection of mBSA into the right knee. Bone marrow chimeras were generated and similarly treated. Joints were harvested and prepared for histologic assessment. T cell responses were verified by cytokine and proliferation responses, and relative immunoglobulin responses were measured by enzyme-linked immunosorbent assay. Cytokine messenger RNA expression levels were measured by quantitative polymerase chain reaction analysis. Thioglycollate-elicited and zymosan A-elicited macrophage recruitment was tested in vivo, and cell migration was tested in vitro. The peptide inhibitor D-JNKi was injected daily starting 4 days after intraarticular injection of mBSA into wild-type (WT) mice, and inflammation was scored histologically. RESULTS: JNK-1-deficient, but not JNK-2-deficient, mice had a reduction in inflammatory cell infiltration and joint damage. This effect was primarily restricted to hematopoietic cells, but B and T cell responses were preserved in mBSA-injected mice. JNK-1-deficient macrophages produced cytokines and chemokines at a level comparable to that in their WT counterparts. However, macrophage migration was impaired in vivo and in vitro. Targeting JNK with the peptide inhibitor D-JNKi dramatically reduced inflammation and joint destruction in WT mice. CONCLUSION: AIA is dependent on JNK-1, but not JNK-2. JNK-1 is a promising molecular target for reducing autoimmune inflammation, since its inhibition impairs macrophage migration.

N-WASP has the ability to compensate for the loss of WASP in macrophage podosome formation and chemotaxis.

Wiskott-Aldrich syndrome protein (WASP) and its homologue neural-WASP (N-WASP) are nucleation promoting factors that integrate receptor signaling with actin cytoskeleton rearrangement. While hematopoietic cells express both WASP and N-WASP, WASP deficiency results in altered cell morphology, loss of podosomes and defective chemotaxis. It was determined that cells from a mouse derived monocyte/macrophage cell line and primary cells of myeloid lineage expressed approximately 15-fold higher levels of WASP relative to N-WASP. To test whether N-WASP can compensate for the loss of WASP and restore actin cytoskeleton integrity, N-WASP was overexpressed in macrophages, in which endogenous WASP expression was reduced by short hairpin RNA (shWASP cells). Many of the defects associated with the loss of WASP, such as podosome-dependent matrix degradation and chemotaxis were corrected when N-WASP was expressed at equimolar level to that of the wild-type WASP. Furthermore, the ability of N-WASP to partially compensate for the loss of WASP may be physiologically relevant since activated murine WASP-deficient peritoneal macrophages, which show enhanced N-WASP expression, also show an increase in matrix degradation. Our study suggests that expression levels of WASP and N-WASP may influence their roles in actin cytoskeleton rearrangement and shed light to the complex intertwining roles WASP and N-WASP play in macrophages.

Strain and model dependent differences in inflammatory cell recruitment in mice.

OBJECTIVE AND DESIGN: The objective of this study was to determine genetic differences in inflammation in these distinct inbred mouse strains. METHODS: Peritoneal leukocyte recruitment, matrix metalloproteinases and cytokines were quantified in A/J, 129/svJ, C57BL/6J, using thioglycollate or biomaterial implants as inflammatory stimuli. RESULTS: In response to thioglycollate, A/J had significant decreases compared to C57BL/6J in both neutrophil (86 %) and macrophage (62 %) recruitment, and 129/svJ had a significant (43 %) decrease compared to C57BL/6J in macrophage recruitment. The reduced leukocyte recruitment corresponded to reduced matrix metalloproteinase-9. In the bioimplant model, 129/svJ had a 2-fold increase in neutrophil and macrophage recruitment compared to C57BL/6J, and the increased leukocyte recruitment corresponded to elevated cytokines, monocyte inhibitory protein-2 and monocyte chemoattractant protein-1, in the lavage compared to the values for C57BL/6J. CONCLUSION: Not only was leukocyte recruitment strain dependent, but the three strains had marked differences in metalloproteinases and cytokine response. In addition, there were model specific differences in the metalloproteinase and cytokine response to the two inflammatory stimuli. Thus, inflammatory cell recruitment is genetically determined and stimulus specific and may determine the susceptibility to complex diseases.

In vivo responses to vaccination with Mage-b, GM-CSF and thioglycollate in a highly metastatic mouse breast tumor model, 4T1.

Metastatic breast cancer is an important contributor to morbidity and mortality. Hence, new therapies are needed that target breast cancer metastases. Here, we focus on Mage-b as a possible vaccine target to prevent the development of breast cancer metastases, through activation of Mage-b-specific cytotoxic T lymphocytes (CTL). The syngeneic cell line 4T1, highly expressing Mage-b, was used as a pre-clinical metastatic mouse breast tumor model. BALB/c mice received three preventive intraperitoneal immunizations with Mage-b DNA vaccine mixed with plasmid DNA, secreting granulocyte-macrophage colony stimulating factor (GM-CSF). In addition, antigen-presenting cells were more efficiently recruited to the peritoneal cavity by the injection of thioglycollate broth (TGB), prior to each immunization. Immunization with Mage-b/GM-CSF/TGB significantly reduced the number of metastases by 67% compared to the saline/GM-CSF/TGB and by 69% compared to the vector control/GM-CSF/TGB. Also, tumor growth was significantly reduced by 45% in mice vaccinated with Mage-b/GM-CSF/TGB compared to the saline/ GM-CSF/TGB and by 47% compared to the control vector/ GM-CSF/TGB group. In vivo, the number of CD8 T cells significantly increased in the primary tumors and metastases of mice vaccinated with Mage-b/GM-CSF/TGB compared to the saline/GM-CSF/TGB and the control vector/ GM-CSF/TGB group, while the number of CD4 T cells significantly decreased. The combination of Mage-b, GM-CSF and TGB did not only induce significantly higher levels of IFNgamma in the lymph nodes of vaccinated compared to control mice, but also induced significantly higher expression levels of Fas-ligand (FasL) in the primary tumors (expressing Fas protein constitutively), compared to the control mice. Whether the interaction between Fas and FasL may have contributed to the smaller tumors needs to be further analyzed.

Mechanisms of tolerance induced by TGF beta-treated APC: CD4 regulatory T cells prevent the induction of the immune response possibly through a mechanism involving TGF beta.

Transforming growth factor beta (TGF beta)-treated antigen-presenting cells (APC) pulsed with antigen induce tolerance in mice, i.e. inhibition of IFN-gamma production and delayed type hypersensitivity response. Although evidence suggests that regulatory T cells are involved, their mechanism of action is currently unknown and is the subject of the present study. Both CD4 and CD8 splenic T cells from mice injected i.v. with adherent thioglycolate-elicited peritoneal exudate cells cultured with TGF beta(2) and antigen (TGF beta-treated APC) transferred tolerance to naive recipients. Interestingly, TGF beta-treated APC from class II knockout mice were unable to induce tolerance in wild-type mice, whereas wild-type TGF beta-treated APC could induce tolerance in CD8 knockout mice. TGF beta was detected in cultures of lymphoid cells from mice injected with TGF beta-treated APC, and treatment with anti-TGF beta antibody in vivo impaired tolerance induction. TGF beta appeared to be involved in both the development of CD4 regulatory T cells and the effector function of the CD4 regulatory T cells. In summary, the important findings in this study are that CD4, and not CD8, regulatory T cells are required for tolerance induced by TGF beta-treated APC in naive mice, and tolerance appears to be mediated by a mechanism involving TGF beta.

Kinetics of the phenotype and function of murine peritoneal macrophages following acute inflammation.

This study was undertaken to have a better understanding for the process and the underlying mechanisms to limit macrophage activation and population of activated macrophages. A comprehensive kinetics of cytokine production was performed in murine peritoneal macrophages recovered from Balb/c mice at various time during the course of an intraperitoneal injection with thioglycollate (TG). The expression of cell surface molecules such as MHC-I, MHC-II, B7-1 and B7-2 of these macrophages were also determined by flow cytometry. The present findings of our research suggested that the population of activated macrophages and the activation of macrophages (including cytokines production and expression of cell surface functional molecules) were strictly controlled during inflammation process. This is one of the important mechanisms to retain the host homeostasis.

Increased vaccination efficiency with apoptotic cells by silica-induced, dendritic-like cells.

We have demonstrated previously the ability of apoptotic cells to prime a functional immune response using an i.p. vaccination protocol with apoptotic cells and interleukin 2, before injecting a lethal dose of tumor cells into syngeneic rats. This protocol resulted in a survival rate of 33%. To elucidate the nature and the activity of the phagocytes involved in the clearance of apoptotic cells in vivo, we modulated the peritoneal cavity environment by administrating either thioglycollate or silica i.p. before injecting the apoptotic cells. Our results showed that thioglycollate abrogated vaccination efficiency, because none of the rats survived under these conditions. In fact, thioglycollate treatment induced a massive recruitment and activation of inflammatory macrophages that efficiently engulfed apoptotic cells, bypassing induction of specific immune responses. In contrast, silica treatment enhanced the vaccination efficiency of apoptotic cells plus interleukin 2 up to 66%. We distinguished a population of dendrite-like cells among the cells derived from the silica-treated peritoneal cavity both by their phenotype (MHC II(+)/CD80(+)/CD86(+)) and by their ability to induce the proliferation of allogeneic T cells in a mixed leukocyte reaction. Our results demonstrate the different roles of macrophages and dendritic-like cells in the physiological clearance of dead tumor cells and their implication in the design of immunomodulating vaccines.

Generation and analysis of mice lacking the chemokine fractalkine.

Fractalkine (CX(3)CL1) is the first described chemokine that can exist either as a soluble protein or as a membrane-bound molecule. Both forms of fractalkine can mediate adhesion of cells expressing its receptor, CX(3)CR1. This activity, together with its expression on endothelial cells, suggests that fractalkine might mediate adhesion of leukocytes to the endothelium during inflammation. Fractalkine is also highly expressed in neurons, and its receptor, CX(3)CR1, is expressed on glial cells. To determine the biologic role of fractalkine, we used targeted gene disruption to generate fractalkine-deficient mice. These mice did not exhibit overt behavioral abnormalities, and histologic analysis of their brains did not reveal any gross changes compared to wild-type mice. In addition, these mice had normal hematologic profiles except for a decrease in the number of blood leukocytes expressing the cell surface marker F4/80. The cellular composition of their lymph nodes did not differ significantly from that of wild-type mice. Similarly, the responses of fractalkine(-/-) mice to a variety of inflammatory stimuli were indistinguishable from those of wild-type mice.

BLTR mediates leukotriene B(4)-induced chemotaxis and adhesion and plays a dominant role in eosinophil accumulation in a murine model of peritonitis.

Leukotriene B(4) (LTB(4)) is a potent chemoattractant active on multiple leukocytes, including neutrophils, macrophages, and eosinophils, and is implicated in the pathogenesis of a variety of inflammatory processes. A seven transmembrane-spanning, G protein-coupled receptor, called BLTR (LTB(4) receptor), has recently been identified as an LTB(4) receptor. To determine if BLTR is the sole receptor mediating LTB(4)-induced leukocyte activation and to determine the role of LTB(4) and BLTR in regulating leukocyte function in inflammation in vivo, we generated a BLTR-deficient mouse by targeted gene disruption. This mouse reveals that BLTR alone is responsible for LTB(4)-mediated leukocyte calcium flux, chemotaxis, and firm adhesion to endothelium in vivo. Furthermore, despite the apparent functional redundancy with other chemoattractant-receptor pairs in vitro, LTB(4) and BLTR play an important role in the recruitment and/or retention of leukocytes, particularly eosinophils, to the inflamed peritoneum in vivo. These studies demonstrate that BLTR is the key receptor that mediates LTB(4)-induced leukocyte activation and establishes a model to decipher the functional roles of BLTR and LTB(4) in vivo.

Procoagulant and anticoagulant activities of resident and inflammatory peritoneal cells.

OBJECTIVE AND DESIGN: In the present study, the involvement of both procoagulants and anticoagulants from adherent peritoneal cells in an inflammatory model was investigated. MATERIAL AND TREATMENT: Mice were injected with thioglycollate broth or lipopolysaccharide (LPS). METHODS: Cells were harvested by peritoneal lavage. Adherent peritoneal cells were cultured further +/- LPS. Subsequently, their ability to activate either Factor X or prothrombin, and their ability to inactivate thrombin through a thrombin-degrading mast cell chymase, was assayed. RESULTS: Inflammatory cells expressed reduced amounts of thrombin-inactivating activity as compared with control resident peritoneal cells. Both resident and inflammatory cells expressed potent prothrombinase activities. Further stimulation of the various cellular populations with LPS in vitro had very little effect on the prothrombinase and thrombin-inactivating activities, bud had a strong stimulatory effect on the Factor X-activating activities.

Macrophage activity in resistant and susceptible mouse strains infected with Mycobacterium lepraemurium.

The level of activation of peritoneal macrophages following subcutaneous inoculation of resistant (C57BL) and susceptible (BALB/c) mice was assessed by monitoring superoxide anion and hydrogen peroxide production and also tumour cell cytostasis. The level of systemic macrophage activation appeared to correlate with bacterial load, rather than resistance to infection. It was observed that the more susceptible (BALB/c) strain developed higher and more sustained levels of systemic macrophage activation, whereas the more resistant (C57BL) strain showed only low transient levels of macrophage activation. In contrast, in vivo challenge of subcutaneously infected C57BL mice, via the intra-peritoneal route, with heat-killed Mycobacterium lepraemurium and thioglycollate resulted in a high level of macrophage activation compared with similarly treated uninfected mice. Similar treatment of susceptible BALB/c mice, however, did not result in enhanced macrophage activation. It was also observed that high levels of macrophage activation occurred in T-cell deprived C57BL mice following infection with M. lepraemurium.

Regulation of the antibody response by the acute phase reactant: mouse serum amyloid P-component (SAP).

Serum amyloid P-component (SAP) is the major acute phase reactant (APR) of mice. Purified mouse SAP at 0.1 to 10.0 micrograms/ml selectively suppressed the secondary in vitro IgG antibody plaque-forming cell (PFC) response to the T-dependent antigen TNP-KLH but not to the T-independent antigens TNP-LPS and DNP-Lys-Ficoll. The suppression was antigen nonspecific. The mechanism of suppression occurred primarily through the activation of Lyt-1+, I-J+ suppressor-inducer cells, which in turn activated a Lyt-2+ suppressor T-cell population. The activity of preexisting, antigen-specific Lyt-2+ suppressor T cells was not influenced by SAP. The antigen-nonspecific suppressor T cells generated by SAP were sensitive to cyclophosphamide. Removal of SAP from the culture fluid with rabbit anti-Mo SAP antibody or agarose beads abrogated the suppression. Pentraxin proteins closely related to mouse SAP, such as human SAP and hamster female protein (FP), also displayed immunoregulatory activity of the antibody response by the same cellular mechanism. The results suggest that SAP regulates antibody responses by the activation of suppressor-inducer T cells and that the regulation of the antibody response during the acute stage of inflammation may occur via SAP.

Delayed type hypersensitivity against semliki forest virus in mice: local transfer of delayed type hypersensitivity with thioglycollate-induced peritoneal exudate cells.

In mice, a strong delayed type hypersensitivity (DH) without detectable neutralizing antibodies in serum could be obtained after intracutaneous injection of inactivated Semliki Forest virus (SFV) mixed with the adjuvant dimethyl dioctadecyl ammonium bromide (DDA). Thioglycollate-induced peritoneal exudate cells (PEC) from these mice were highly effective in passive transfer of DH against SFV locally in footpads of naive recipient mice. DH reactions were measured with a footpad swelling test. By contrast, either immune PEC from nonstimulated peritoneal cavities or thioglycollate-induced PEC from mice which had developed neutralizing antibodies were unable to transfer DH passively.