C-fms Promoter Research Articles

A novel mouse model of systemic hypertension by neprilysin overexpression in myeloid cells

There have been many hypertension mouse models created by transgenesis. The transgenesis technologies to create hypertensive mouse models mostly target the renin-angiotensin system (RAS). Here, we show that enhanced expression of neprilysin driven by the myeloid-specific c-fms promoter causes systemic hypertension in mice. [Method] Transgenic mice, termed Nep mice, were created with a construct containing the c-fms promoter and the neprilysin gene (MME). Blood pressure was measured by tail-cuff. Circulating vasoactive substances were analyzed by ELISAs. [Results] Nep mice showed higher systolic blood pressure compared with WT mice (117.0±1.75 vs. 107.1±2.373 mmHg, p=0.0018). The sodium concentration in a 24hr urine was significantly lower in Nep mice (0.2093±0.0039 vs. 0.2445±0.0054 μmol/μl urine, p=0.0004). The concentration of circulating neprilysin was significantly higher in Nep mice (3085±750.5 vs. 1098±197.1 pg/ml, p=0.0283). Notably, the plasma concentration of ANP showed no significant difference between Nep and WT mice although hypertension usually causes an ANP increase. [Conclusion] Overexpression of neprilysin in monocytes causes systemic hypertension associated with sodium and water retention. These mice might be useful for studying the pathologies of heart diseases and hypertension due to volume overload, and in addition, other neprilysin related diseases like Alzheimer’s disease to investigate the interaction with immune cells and beta-amyloid which is cleaved by neprilysin. NIH R01 AI164519 (KEB, P.I.) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Development of a new macrophage-specific TRAP mouse (MacTRAP) and definition of the renal macrophage translational signature

Tissue macrophages play an important role in organ homeostasis, immunity and the pathogenesis of various inflammation-driven diseases. One major challenge has been to selectively study resident macrophages in highly heterogeneous organs such as kidney. To address this problem, we adopted a Translational Ribosome Affinity Purification (TRAP)- approach and designed a transgene that expresses an eGFP-tagged ribosomal protein (L10a) under the control of the macrophage-specific c-fms promoter to generate c-fms-eGFP-L10a transgenic mice (MacTRAP). Rigorous characterization found no gross abnormalities in MacTRAP mice and confirmed transgene expression across various organs. Immunohistological analyses of MacTRAP kidneys identified eGFP-L10a expressing cells in the tubulointerstitial compartment which stained positive for macrophage marker F4/80. Inflammatory challenge led to robust eGFP-L10a upregulation in kidney, confirming MacTRAP responsiveness in vivo. We successfully extracted macrophage-specific polysomal RNA from MacTRAP kidneys and conducted RNA sequencing followed by bioinformatical analyses, hereby establishing a comprehensive and unique in vivo gene expression and pathway signature of resident renal macrophages. In summary, we created, validated and applied a new, responsive macrophage-specific TRAP mouse line, defining the translational profile of renal macrophages and dendritic cells. This new tool may be of great value for the study of macrophage biology in different organs and various models of injury and disease.

Myeloid cell responsiveness to interferon-gamma is sufficient for initial resistance to Listeria monocytogenes

The type II interferon (IFNγ) promotes resistance to intracellular pathogens. Most immune and somatic cells also express the IFNγ receptor (IFNGR) and respond to IFNγ. While myeloid cell have been implicated as important targets of IFNγ, it remains unknown if IFNγ signaling to myeloid cell types suffices for resistance to infection. Here, we addressed this question by generating mice in which IFNGR1 is selectively expressed by myeloid cells. These “MSGR1” (myeloid selective IFNGR1) mice express an epitope-tagged Ifngr1 transgene (fGR1) from the myeloid-specific c-fms promoter in a background lacking endogenous Ifngr1. IFNGR staining was selectively observed on myeloid cells in the MSGR1 mice and correlated with responsiveness of these cells to IFNγ. During systemic infection by the bacterium Listeria monocytogenes, activation marker staining was comparable on monocytes from MSGR1 and control B6 mice. Bacterial burdens and survival were also equivalent in MSGR1 and wildtype B6 animals at a timepoint when B6.Ifngr1−/− mice began to succumb. These data confirm that activation of inflammatory monocytes and neutrophils is a key mechanism by which IFNγ promotes innate anti-bacterial immunity and suggest that IFNγ targeting of myeloid cells is largely sufficient to mediate protection against systemic L. monocytogenes.

Constitutive AKT Activity Predisposes Lung Fibrosis by Regulating Macrophage, Myofibroblast and Fibrocyte Recruitment and Changes in Autophagy.

The etiology and pathogenesis of pulmonary fibrosis is poorly understood. We and others reported that M-CSF/CSF-1, M-CSF-R and downstream AKT activation plays an important role in lung fibrosis in mice models and in IPF patients. To understand potential molecular pathways used by M-CSF-R activation to direct lung fibrosis, we used a novel transgenic mouse model that expresses a constitutively-active form of AKT, myristoylated AKT (Myr-Akt), driven by the c-fms (M-CSF-R) promoter. We were particularly interested in the basal immune state of the lungs of these Myr-Akt mice to assess M-CSF-R-related priming for lung fibrosis. In support of a priming effect, macrophages isolated from the lungs of unchallenged Myr-Akt mice displayed an M2-tropism, enhanced co-expression of M-CSF-R and α-SMA, reduced autophagy reflected by reduced expression of the key autophagy genes Beclin-1, MAP1-Lc3a(Lc3a), and MAP1-Lc3b(Lc3b), and increased p62/STSQM1 expression compared with littermate WT mice. Furthermore, Myr-Akt mice had more basal circulating fibrocytes than WT mice. Lastly, upon bleomycin challenge, Myr-Akt mice showed enhanced collagen deposition, increased F4/80+ and CD45+ cells, reduced autophagy genes Beclin-1, Lc3a, and Lc3b expression, and a shorter life-span than WT littermates. These data provide support that M-CSF-R/AKT activation may have a priming effect which can predispose lung tissue to pulmonary fibrosis.

HIV-1 Nef expression in microglia disrupts dopaminergic and immune functions with associated mania-like behaviors

Neuropsychiatric disorders during HIV/AIDS are common although the contribution of HIV-1 infection within the brain, and in particular individual HIV-1 proteins, to the development of these brain disorders is unknown. Herein, an in vivo transgenic mouse model was generated in which the HIV-1 Nef protein was expressed in microglia cells, permitting investigation of neurobehavioral phenotypes and associated cellular and molecular properties. Transgenic (Tg) mice that expressed full length HIV-1 nef under the control of the c-fms promoter and wildtype (Wt) littermates were investigated using different measures of neurobehavioral performance including locomotory, forced swim (FST), elevated plus maze (EPM) and T-maze tests. Host gene and transgene expression were assessed by RT-PCR, immunoblotting, enzymatic activity and immunohistochemistry. Biogenic amine levels were measured by HPLC with electrochemical detection. Tg animals exhibited Nef expression in brain microglia and cultured macrophages. Tg males displayed hyperactive behaviors including augmented locomotor activity, decreased immobility in the FST and increased open-arm EPM exploration compared to Wt littermates (p<0.05). Tg animals showed increased CCL2 expression with concurrent IFN-α suppression in striatum compared with Wt littermates (p<0.05). Dopamine levels, MAO activity and the dopamine transporter (DAT) expression were reduced in the striatum of Tg animals (p<0.05). HIV-1 Nef expression in microglia induced CCL2 expression together with disrupting striatal dopaminergic transmission, resulting in hyperactive behaviors which are observed in mania and other psychiatric comorbidities among HIV-infected persons. These findings emphasize the selective effects of individual viral proteins in the brain and their participation in neuropathogenesis.

Angiotensin-converting enzyme overexpression in myelomonocytes prevents Alzheimer’s-like cognitive decline

Cognitive decline in patients with Alzheimer's disease (AD) is associated with elevated brain levels of amyloid β protein (Aβ), particularly neurotoxic Aβ(1-42). Angiotensin-converting enzyme (ACE) can degrade Aβ(1-42), and ACE overexpression in myelomonocytic cells enhances their immune function. To examine the effect of targeted ACE overexpression on AD, we crossed ACE(10/10) mice, which overexpress ACE in myelomonocytes using the c-fms promoter, with the transgenic APP(SWE)/PS1(ΔE9) mouse model of AD (AD⁺). Evaluation of brain tissue from these AD⁺ACE(10/10) mice at 7 and 13 months revealed that levels of both soluble and insoluble brain Aβ(1-42) were reduced compared with those in AD⁺ mice. Furthermore, both plaque burden and astrogliosis were drastically reduced. Administration of the ACE inhibitor ramipril increased Aβ levels in AD⁺ACE(10/10) mice compared with the levels induced by the ACE-independent vasodilator hydralazine. Overall, AD⁺ACE(10/10) mice had less brain-infiltrating cells, consistent with reduced AD-associated pathology, though ACE-overexpressing macrophages were abundant around and engulfing Aβ plaques. At 11 and 12 months of age, the AD⁺ACE(10/WT) and AD⁺ACE(10/10) mice were virtually equivalent to non-AD mice in cognitive ability, as assessed by maze-based behavioral tests. Our data demonstrate that an enhanced immune response, coupled with increased myelomonocytic expression of catalytically active ACE, prevents cognitive decline in a murine model of AD.

25-Hydroxycholesterol Activates the Integrated Stress Response to Reprogram Transcription and Translation in Macrophages

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.

NF-kappaB activation within macrophages leads to an anti-tumor phenotype in a mammary tumor lung metastasis model

IntroductionMetastasis from primary tumor to the lungs is a major cause of the mortality associated with breast cancer. Both immune and inflammatory responses impact whether circulating mammary tumor cells successfully colonize the lungs leading to established metastases. Nuclear factor -kappaB (NF-κB) transcription factors regulate both immune and inflammatory responses mediated in part by the activities of macrophages. Therefore, NF-κB activity specifically within macrophages may be a critical determinant of whether circulating tumor cells successfully colonize the lungs.MethodsTo investigate NF-κB signaling within macrophages during metastasis, we developed novel inducible transgenic models which target expression of the reverse tetracycline transactivator (rtTA) to macrophages using the cfms promoter in combination with inducible transgenics that express either an activator (cIKK2) or an inhibitor (IκBα-DN). Doxycyline treatment led to activation or inhibition of NF-κB within macrophages. We used a tail vein metastasis model with mammary tumor cell lines established from MMTV-Polyoma Middle T-Antigen-derived tumors to investigate the effects of modulating NF-κB in macrophages during different temporal windows of the metastatic process.ResultsWe found that activation of NF-κB in macrophages during seeding leads to a reduction in lung metastases. The mechanism involved expression of inflammatory cytokines and reactive oxygen species, leading to apoptosis of tumor cells and preventing seeding in the lung. Activation of NF-κB within macrophages after the seeding phase has no significant impact on establishment of metastases.ConclusionsOur results have identified a brief, defined window in which activation of NF-κB has significant anti-metastatic effects and inhibition of NF-κB results in a worse outcome.

Angiotensin-converting Enzyme Overexpression in Mouse Myelomonocytic Cells Augments Resistance to Listeria and Methicillin-resistant Staphylococcus aureus

Gene targeting in ES cells was used to substitute control of angiotensin converting enzyme (ACE) expression from the endogenous promoter to the mouse c-fms promoter. The result is an animal model called ACE 10/10 in which ACE is overexpressed by monocytes, macrophages, and other myelomonocytic lineage cells. To study the immune response of these mice to bacterial infection, we challenged them with Listeria monocytogenes or methicillin-resistant Staphylococcus aureus (MRSA). ACE 10/10 mice have a significantly enhanced immune response to both bacteria in vivo and in vitro. For example, 5 days after Listeria infection, the spleen and liver of ACE 10/10 mice had 8.0- and 5.2-fold less bacteria than wild type mice (WT). In a model of MRSA skin infection, ACE 10/10 mice had 50-fold less bacteria than WT mice. Histologic examination showed a prominent infiltrate of ACE-positive mononuclear cells in the skin lesions from ACE 10/10. Increased bacterial resistance in ACE 10/10 is directly due to overexpression of ACE, as it is eliminated by an ACE inhibitor. Critical to increased immunity in ACE 10/10 is the overexpression of iNOS and reactive nitrogen intermediates, as inhibition of iNOS by the inhibitor 1400W eliminated all in vitro and in vivo differences in innate bacterial resistance between ACE 10/10 and WT mice. Increased resistance to MRSA was transferable by bone marrow transplantation. The overexpression of ACE and iNOS by myelomonocytic cells substantially boosts innate immunity and may represent a new means to address serious bacterial infections.

Abstract 1352: Macrophage specific regulation reveals both pro- and anti-tumor effects of NF-kappaB during mammary tumor progression

Abstract An increased level of macrophages in human tumors is associated with a poorer prognosis. The transcription factor Nuclear Factor-kappaB (NF-kappaB) is an important regulator of gene expression within macrophages. Recent studies point to a pro-tumorigenic role for NF-kappaB activity in macrophages; however NF-kappaB also regulates expression of genes such as inducible nitric oxide synthase which are associated with anti-tumor effects. We believe that NF-kappaB activity in macrophages can have both anti- and pro-tumor effects depending on tumor stage. We have developed novel murine models to determine the effects of modulation of NF-kappaB activity specifically within macrophages during primary mammary tumor growth and metastasis. We have used the cfms promoter to specifically express the reverse tetracycline transactivator (rtTA) protein in macrophages. The rtTA can drive tet operon regulated gene expression in the presence of doxycyline (dox). We have used this with a dominant negative IkappaBalpha to inhibit NF-kappaB signaling in the Polyoma mouse mammary tumor model, this model is termed DNFP. We have also used the cfms-rtTA in combination with a constitutively active IKK2 transgene to activate NF-kappaB signaling, this model is termed IKFM. When NF-kappaB is inhibited in macrophages during primary tumor growth in our DNFP model there is a reduction in primary tumor growth accompanied by a reduced level of lung metastasis. This data suggests that NF-kappaB in macrophages is acting in a pro-tumor manner, in agreement with recently published data. In contrast, we have found that activation of NF-kappaB in macrophages by treating IKFM mice with dox for 1 week before and during two weeks following tail vein injection of polyoma mammary tumor cells leads to a significant reduction in surface lung tumor formation. In additional studies, IKFM mice were pretreated with dox for 1 week but treatment was stopped two days after tumor injection. A similar reduction in tumor number was observed, suggesting an inhibition of cell seeding. Real time PCR analysis of lungs from mice with activation of NF-kappaB in macrophages suggests an anti-tumor “M1” macrophage phenotype. In summary, our data indicates that NF-kappaB activity in macrophages can have both anti- and pro-tumor effects dependent on tumor stage. This data would suggest that cell specificity and timing of treatment are important considerations with regards to the use of NF-kappaB inhibitors in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1352.

Pulmonary Inflammation Triggered by Ricin Toxin Requires Macrophages and IL-1 Signaling

Ricin is a potent ribotoxin considered to be a potentially dangerous bioterrorist agent due to its wide availability and the possibility of aerosol delivery to human populations. Studies in rodents and nonhuman primates have demonstrated that ricin delivered to the pulmonary system leads to acute lung injury and symptoms resembling acute respiratory distress syndrome. Increasing evidence suggests that the inflammatory effects triggered by ricin are responsible for its lethality. We demonstrated previously that ricin administered to the lungs of mice causes death of pulmonary macrophages and the release of proinflammatory cytokines, suggesting macrophages may be a primary target of ricin. Here we examined the requirement for macrophages in the development of ricin-mediated pulmonary inflammation by employing transgenic (MAFIA) mice that express an inducible gene driven by the c-fms promoter for Fas-mediated apoptosis of macrophages upon injection of a synthetic dimerizer, AP20187. Administration of aerosolized ricin to macrophage-depleted mice led to reduced inflammatory responses, including recruitment of neutrophils, expression of proinflammatory transcripts, and microvascular permeability. When compared with control mice treated with ricin, macrophage-depleted mice treated with ricin displayed a reduction in pulmonary IL-1beta. Employing mice deficient in IL-1, we found that ricin-induced inflammatory responses were suppressed, including neutrophilia. Neutrophilia could be restored by co-administering ricin and exogenous IL-1beta to IL-1alpha/beta(-/-) mice. Furthermore, IL1Ra/anakinra cotreatment inhibited ricin-mediated inflammatory responses, including recruitment of neutrophils, expression of proinflammatory genes, and histopathology. These data suggest a central role for macrophages and IL-1 signaling in the inflammatory process triggered by ricin.

Bone Marrow Chimeras and c-fms Conditional Ablation (Mafia) Mice Reveal an Essential Role for Resident Myeloid Cells in Lipopolysaccharide/TLR4-Induced Corneal Inflammation

The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4(-/-) mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)(+)/C57BL/6 or eGFP(+)/TLR4(-/-) mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4(-/-) mice reconstituted with C57BL/6, but not TLR4(-/-) bone marrow cells donor cells were found to cause infiltration of eGFP(+) cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP(+) cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1alpha production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation.

Bone Marrow Monocyte/Macrophages Provide Signals Necessary for Osteoblast Maintenance: Potential Role for Insulin-Like Growth Factor Signaling

There is considerable evidence showing that osteoblast lineage cells play a key role in regulating hematopoiesis. Less clear is the contribution of hematopoietic cells to osteoblast maintenance. We and others previously showed that treatment with granulocyte-colony stimulating factor (G-CSF) results in a striking loss of osteoblast number and function. Bone marrow transplantation experiments demonstrated that G-CSF does not directly suppress osteoblast function but acts through a transplantable hematopoietic intermediary. Earlier work from our lab suggested that mature neutrophils and B- and T-lymphocytes are dispensable for G-CSF-induced osteoblast loss. We therefore hypothesized that bone marrow monocytes play a role in regulating osteoblasts both during G-CSF treatment and at steady state. To begin to test this hypothesis, primary murine bone marrow stromal cultures—consisting of both adherent macrophages and mesenchymal-lineage stromal cells—were established and cultured three weeks in osteogenic media. Removal of macrophages from these cultures resulted in a striking reduction in osteoblast differentiation, as evidenced by reduced alkaline phosphatase and sharply reduced expression of osteocalcin (92% reduction, p<.0001, n=6), a mature osteoblast marker. To investigate whether bone marrow monocytes play a role in supporting osteoblasts in vivo, we made use of transgenic mice expressing an inducible suicide gene in the monocyte compartment under control of the c-fms promoter. Induction of the suicide gene by administration of an exogenous ligand resulted in efficient deletion of monocytes and macrophages from the bone marrow and peripheral blood. Histomorphometry of long bones showed a striking decrease in osteoblast number in monocyte-ablated mice compared to controls, and osteocalcin mRNA decreased to nearly undetectable levels (n=7 each group). The loss of osteoblasts after monocyte ablation does not result from bystander killing, as other boneassociated cells such as osteoclasts and osteoprogenitors are not decreased. Monocyte ablation and osteoblast loss were associated with a 15-fold mobilization of hematopoietic progenitors (n=5 each group), comparable to levels attained after G-CSF treatment. Taken together, these in vitro and in vivo findings suggest that monocytes provide factors necessary for osteoblast survival or development and raise the possibility that G-CSF administration modulates these factors. As a first step toward identifying these factors, expression profiling of monocytes harvested from the bone marrow of untreated or G-CSF treated mice was performed. These data show that insulin-like growth factor 1 (IGF1) is produced at high levels in resting bone marrow monocytes and is downregulated more than 2.5-fold by G-CSF. Moreover, expression of IGF binding protein 6 (IGFBP6), an IGF binding-protein family member that negatively regulates IGF signaling, is upregulated more than 6-fold in monocytes by G-CSF. Quantitative real time PCR confirmed that IGF- 1 mRNA is reduced >11-fold in bone marrow from monocyte-ablated transgenic mice (p<.05, n=2–3 each group) and >7-fold in wild type mice treated with G-CSF (p<.05, n=3 each group). In addition, IGFBP6 expression was increased ~100-fold in bone marrow during G-CSF treatment. Fractionation of hematopoietic cell populations confirmed that IGFBP6 mRNA and protein is induced in monocyte and granulocyte populations in the bone marrow during G-CSF treatment. Collectively, these data support a model in which monocytes provide signals required for osteoblast maintenance in adult bone marrow. IGF1 signaling is known to play a vital role in osteoblast development and maintenance. Thus, these data also suggest that G-CSF may modulate osteoblast number and function (and secondarily induce hematopoietic stem cell mobilization) through alteration of IGF1 and IGFBP6 expression by monocytes. Studies are underway to directly assess the affect of IGFBP6 on osteoblast survival and differentiation.

Mice with Enhanced Macrophage Angiotensin-Converting Enzyme Are Resistant to Melanoma

Angiotensin-converting enzyme (ACE) is a peptidase responsible for the cleavage of angiotensin I and several other peptides. Here, gene targeting was used to switch control of the ACE locus from the endogenous promoter to the macrophage-specific c-fms promoter. Challenge of these mice, called ACE 10/10, with the aggressive mouse melanoma cell line B16 showed that they are remarkably resistant to tumor growth. Tumor resistance was seen after challenge with different melanoma cell lines and in mice with different genetic backgrounds. Histological study of the tumors that did grow in ACE 10/10 mice showed an enhanced inflammatory response. ACE 10/10 mice had increased numbers of tumor epitope-specific CD8(+) T cells after challenge with melanoma or lymphoma. ACE 10/10 macrophages showed increased production of interleukin-12 and nitric oxide but reduced interleukin-10. Engraftment of wild-type mice with ACE 10/10 bone marrow transferred B16 tumor resistance. Injection of B16 tumors with ACE 10/10 macrophages also reduced tumor growth. ACE 10/10 mice may define a new means of enhancing the immune response, which may be potentially useful in several human clinical situations.

Characterisation and trophic functions of murine embryonic macrophages based upon the use of a Csf1r–EGFP transgene reporter

All solid organs contain resident monocyte-derived cells that appear early in organogenesis and persist throughout life. These cells are critical for normal development in some organs. Here we report the use of a previously described transgenic line, with EGFP driven by the macrophage-restricted Csf1r ( c-fms) promoter, to image macrophage production and infiltration accompanying organogenesis in many tissues. Using microarray analysis of FACS-isolated EGFP-positive cells, we show that fetal kidney, lung and brain macrophages show similar gene expression profiles irrespective of their tissue of origin. EGFP-positive cells appeared in the renal interstitium from 12 days post coitum, prior to nephrogenesis, and maintain a close apposition to renal tubules postnatally. CSF-1 added to embryonic kidney explants increased overall renal growth and ureteric bud branching. Expression profiling of tissue macrophages and of CSF-1-treated explants showed evidence of the alternate, pro-proliferative (M2) activation profile, including expression of macrophage mannose receptor (CD206), macrophage scavenger receptor 2 (Msr2), C1q, CD163, selenoprotein P, CCL24 and TREM2. This response has been associated with the trophic role of tumour-associated macrophages. These findings suggest a trophic role of macrophages in embryonic kidney development, which may continue to play a similar role in postnatal repair.

PU.1 and GATA-1, but Not SCL Elicit an Increase in General Transcription Associated with the Lineage Restriction of Multipotent Progenitors.

The lineage commitment of multipotent progenitor cells is coordinated by regulatory interactions between transcription factor programs associated with different lineage fates. Accordingly, mutations which interfere with these programs can block differentiation and contribute to leukemogenesis. A thorough understanding of lineage commitment processes should therefore identify targets and strategies for differentiation therapy. We have compared the effects of transient over-expression of the hematopoietic transcription factors PU.1, GATA-1 and SCL on the response of a multipotent hematopoietic progenitor cell line (FDCPmix) to conditions supporting either self-renewal, myeloid differentiation or erythroid differentiation.Methods: Pure populations of FDCPmix cells productively transfected with transcription factor cDNA/IRES/GFP expression vectors were isolated by FACS sorting and entered directly into liquid media containing either IL-3 (for self-renewal); GM-CSF + G-CSF (for myeloid differentiation) or erythropoietin + hemin (for erythroid differentiation). Semi-solid medium containing IL-3 was used to assess the maintenance of that sub-population of cells which can undergo self-renewal in isolation, and which are assumed to represent the earliest progenitors in an FDCPmix culture. Transcription factor function was confirmed by co-transfection with reporter genes driven by specific target promoters.Results: The yield of FDCPmix cells from self-renewal medium was markedly enhanced by the transient expression of SCL but reduced by either PU.1 or GATA-1. These differences in yield reflect alterations in survival and/or proliferation over the two day period of transgene expression immediately following transfection. As expected, the transient expression of PU.1 severely reduced the potential of multipotent progenitors to respond to erythroid growth factors, while GATA-1 similarly reduced the response to myeloid growth factors, consistent with an antagonistic relationship between myeloid and erythroid transcription factors during lineage restriction. However, while all three transcription factors demonstrated the appropriate activity on their respective target promoters, PU.1 and GATA-1 also increased general transcriptional activity. Transient expression of GATA-1 actually raised the activity of the cfms (PU.1 target) promoter, while PU.1 activated the serpin 2A (GATA-1 target) promoter. Of the transcription factors tested, only PU.1 consistently reduced the frequency of colony forming cells, suggesting that the earliest multipotent progenitors in an FDCPmix population can be recruited to commitment by PU.1 but not by GATA-1.Conclusion: These results are consistent with a multi-step process of lineage commitment in which general transcriptional activity in multipotent progenitors is maintained at a low level, base state. An increase in general transcriptional activity would then be required as an early event in commitment, activating the transcription factor networks subsequently responsible for coordinating lineage restriction.

Dendritic cells: Not just another cell type in the kidney, but a complex immune sentinel network

Dendritic cells (DCs) are crucial for inducing and regulating adaptive immunity. These cells also exist in the kidney, where, however, their function had been unknown. A study by Soos et al. now demonstrates that renal DCs form an intricate cellular network that continuously surveys the tubulointerstitium, and reveals a previously unrecognized immune sentinel system of the kidney.

Development of Peritoneal Adhesions in Macrophage Depleted Mice

We present a new mouse model for the study of peritoneal adhesions using macrophage Fas-induced apoptosis (Mafia) transgenic mice expressing a Fas-FKBP construct under control of the murine c-fms promoter. Mafia mice allow systemic macrophage depletion by dimerization of Fas with a synthetic dimerizer, AP20187. Results demonstrate that macrophage depletion in Mafia mice induces peritoneal adhesion formation when the peritoneal cavity is also exposed to an irritant. The Mafia mouse model presents a reproducible, non-surgical approach for research in adhesion formation and prevention. Mafia mice were treated with AP20187 using an intravenous (i.v.) or intraperitoneal (i.p.) injection. Control groups included mock-treated Mafia mice and both AP20187 and mock-treated wild type mice. Seven days after treatment, mice were observed for the presence of adhesions. After i.p. injection with AP20187, 76% of Mafia mice developed adhesions whereas none of the mock-treated Mafia or wild-type mice developed adhesions, and only one AP20187-treated wild-type mouse (5.8%) developed a mild adhesion. Mafia mice treated with AP20187 i.v. exhibited macrophage depletion not significantly different than i.p. treated mice, but did not develop adhesions. In contrast, Mafia mice treated with AP20187 i.v. developed adhesions when diluent was also injected into the peritoneal cavity, whereas i.p diluent alone had no effect. Macrophage depletion, combined with a peritoneal irritant, results in peritoneal adhesion formation in transgenic Mafia mice. Macrophages appear to play a protective role in the development and/or repair of peritoneal adhesions.

Subnuclear targeting of Runx1 Is required for synergistic activation of the myeloid specific M‐CSF receptor promoter by PU.1

Many types of acute myelogenous leukemia involve chromosomal translocations that target the C-terminus of Runx1/AML1 transcription factor, a master regulator of hematopoiesis. The C-terminus of Runx1/AML1 that includes the nuclear matrix targeting signal (NMTS) is essential for embryonic development, hematopoiesis, and target gene regulation. During the onset and normal progression of hematopoiesis, several lineage-specific factors such as C/EBPalpha and PU.1 interact with Runx1 to regulate transcription combinatorially. Here we addressed the functional interplay between subnuclear targeting of Runx1 and gene activation during hematopoiesis. Point mutations were generated in the NMTS of the human Runx1 protein and tested for their effect on transcriptional cooperativity with C/EBPalpha and PU.1 at myeloid-specific promoters. We characterized five mutants that do not alter nuclear import, DNA binding or C/EBPalpha-dependent synergistic activation of the target gene promoters. However a critical tyrosine in the NMTS is required for subnuclear targeting and activation of the granulocyte-macrophage colony stimulating factor (GM-CSF) promoter. Furthermore, this point mutation is defective for transcriptional synergism with PU.1 on the macrophage colony stimulating factor (MCSF) receptor c-FMS promoter. Our results indicate that the NMTS region of Runx1 is required for functional interactions with PU.1. Taken together, our findings establish that subnuclear targeting of Runx1 is a critical component of myeloid-specific transcriptional control.

The Colony-Stimulating Factor 1 Receptor Is Expressed on Dendritic Cells during Differentiation and Regulates Their Expansion

The lineage of dendritic cells (DC), and in particular their relationship to monocytes and macrophages, remains obscure. Furthermore, the requirement for the macrophage growth factor CSF-1 during DC homeostasis is unclear. Using a transgenic mouse in which the promoter for the CSF-1R (c-fms) directs the expression of enhanced GFP in cells of the myeloid lineage, we determined that although the c-fms promoter is inactive in DC precursors, it is up-regulated in all DC subsets during differentiation. Furthermore, plasmacytoid DC and all CD11c(high) DC subsets are reduced by 50-70% in CSF-1-deficient osteopetrotic mice, confirming that CSF-1 signaling is required for the optimal differentiation of DC in vivo. These data provide additional evidence that the majority of tissue DC is of myeloid origin during steady state and supports a close relationship between DC and macrophage biology in vivo.