Nico van Rooijen

Liposomes for Specific Depletion of Macrophages from Organs and Tissues

A liposome mediated macrophage suicide approach has been developed, based on the liposome mediated internalization of the small hydrophilic molecule clodronate in macrophages J Leukoc Biol 62:702, 1997. This molecule has a very short half life when released in the circulation, but does not easily cross phospholipid bilayers of liposomes or cell membranes. As a consequence, once ingested by a macrophage in a liposome encapsulated form, it will be accumulated within the cell as soon as the liposomes are digested with the help of its lysosomal phospholipases. At a certain intracellular clodronate concentration, the macrophage is eliminated by apoptosis. Given the fact that, neither the liposomal phospholipids chosen, nor clodronate are toxic to other (non-phagocytic) cells, this method has proven its efficacy and specificity for depletion of macrophage subsets in various organs. In several cases, organ specific depletion can be obtained by choosing the right administration route for the clodronate liposomes.

Neutrophils Transport Antigen from the Dermis to the Bone Marrow, Initiating a Source of Memory CD8+ T Cells

The bone marrow (BM) has been identified as a possible organ for T cell priming, yet the fundamental mechanisms of a polyclonal immune response in the BM remain unknown. We found that after intradermal injection of modified vaccinia Ankara virus, unexpected sources of newly primed polyclonal virus-specific CD8(+), but not CD4(+), T cells were localized in the BM and the draining lymph nodes (dLNs) prior to blood circulation. We identified neutrophils as the virus-carrier cells from the dermis to the BM. In both neutrophil-depleted and Ccr1(-/-) mice, virus-specific BM CD8(+) responses were lost. Myeloid antigen-presenting cells were required for BM CD8(+) T cell priming. A systems biology analysis of dLN and BM virus-specific CD8(+) T cells revealed distinct transcriptional and multifunctional profiles for cells primed in each organ. We provide direct evidence for how antigen is transported to the BM, providing a source of virus-specific memory CD8(+) T cells.

Macrophages direct tumour histology and clinical outcome in a colon cancer model

Macrophages generally constitute a major component of the tumour stroma. Although conventionally considered to be cytotoxic effector cells, macrophages have recently been described as promoters of tumour progression. The present study shows that selective depletion of peritoneal or liver macrophages prior to CC531 tumour cell inoculation resulted in highly differentiated tumours in rats. In contrast, tumours from control rats, in which macrophages are abundantly present, showed a desmoplastic stromal reaction with hallmark features of malignancy, such as neovascularization and matrix remodelling, indicating that the presence of macrophages is associated with malignant histopathological differentiation. Remarkably, macrophage‐depleted rats, bearing highly differentiated tumours, had a worse prognosis, as they displayed a higher tumour load and poorer survival. Thus, while macrophages direct tumours towards malignant tumour histology, their role in anti‐tumour defence is important. The selective inhibition of macrophage functions involved in malignant progression without interfering with cytotoxic ability may therefore identify important new targets for cancer therapy. Copyright

Macrophages promote polycystic kidney disease progression.

Renal M2-like macrophages have critical roles in tissue repair stimulating tubule cell proliferation and, if they remain, fibrosis. M2-like macrophages have also been implicated in promoting cyst expansion in mouse models of autosomal dominant polycystic kidney disease (ADPKD). While renal macrophages have been documented in human ADPKD, there are no studies in autosomal recessive polycystic kidney disease (ARPKD). Here we evaluated the specific phenotype of renal macrophages and their disease-impacting effects on cystic epithelial cells. We found an abundance of M2-like macrophages in the kidneys of patients with either ADPKD or ARPKD and in the cystic kidneys of cpk mice, a model of ARPKD. Renal epithelial cells from either human ADPKD cysts or non-cystic human kidneys promote differentiation of naive macrophages to a distinct M2-like phenotype in culture. Reciprocally, these immune cells stimulate the proliferation of renal tubule cells and microcyst formation in vitro. Further, depletion of macrophages from cpk mice indicated that macrophages contribute to PKD progression regardless of the genetic etiology. Thus M2-like macrophages are two-pronged progression factors in PKD promoting cyst cell proliferation, cyst growth, and fibrosis. Agents that block the emergence of these cells or their effects in the cystic kidney may be effective therapies for slowing PKD progression.

The roles of vitreal macrophages and circulating leukocytes in retinal neovascularization.

PURPOSEnTo analyze the roles of vitreal macrophages and circulating leukocytes in retinal vascular growth.nnnMETHODSnBone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice were transplanted into postnatal day (P)1 mice after irradiation. The mice were exposed to 76% to 78% oxygen (P7-P12), to initiate oxygen-induced retinopathy (OIR). The eyes were collected at P8, P17, and P30, to analyze the engraftment of GFP-positive cells in the retina. GFP-positive peritoneal macrophages, clodronate liposomes, or control liposomes were injected into the eyes at P5 or P12 to examine the effects at P8 or P17. The number of Iba1-positive vitreal macrophages was quantified from histologic sections at P12 and P17.nnnRESULTSnFew transplanted GFP-positive cells were found in the retina at P8 in both wild-type and OIR mice. However, their number increased at P17 during retinal neovascularization in OIR. Most GFP-positive cells were Iba1-positive microglia, which comprised a minority of the total retinal microglia. Intravitreal injection of peritoneal macrophages showed only incidental migration of these cells into the wild-type retinas (P8), whereas the engraftment was more robust, typically around the neovascularization, in OIR mice (P17). Furthermore, native macrophages in the vitreous cavity became fewer (37.7% reduction) during neovascularization in OIR at P17. The selective depletion of vitreal macrophages by clodronate liposomes at P12 reduced retinal neovascularization in OIR mice by 59.0% at P17.nnnCONCLUSIONSnVitreal macrophages are attracted to the site of pathologic angiogenesis triggered by retinal ischemia, where they actively participate in vascular development.

Microglia-Mediated IGF-I Neuroprotection in the rd10 Mouse Model of Retinitis Pigmentosa

PURPOSEnTo characterize the effect of IGF-I in the rd10 mouse model of retinitis pigmentosa at the cellular level, focusing on the role of microglia in the neurodegenerative process.nnnMETHODSnBoth organotypic retinal explants and intravitreal injections were used to assess the effect of IGF-I on photoreceptor cell death in the Pde6b(rd10) mice. Cell death was determined by TUNEL in retinal sections and by ELISA of free nucleosomes in retinal extracts. The number and distribution of microglial cells was visualized by immunolabeling with Cd11b and Iba1 antibodies. Depletion of microglia in culture was achieved by treatment with liposomes containing clodronate.nnnRESULTSnBoth ex vivo and in vivo IGF-I treatment reduced the number of TUNEL-positive nuclei in rd10 mouse retinas. In addition, IGF-I treatment in explants increased the number of microglial cells in the ONL. Depletion of microglia in explants with liposomes containing clodronate diminished the neuroprotective effect of IGF-I but also moderately reduced photoreceptor cell death in rd10 retinas cultured in the absence of IGF-I.nnnCONCLUSIONSnIGF-I is able to attenuate photoreceptor cell death both ex vivo and in vivo in the rd10 mouse retina. Microglia is required for the neuroprotective effect of IGF-I in the dystrophic retina. In addition, microglial cells play a detrimental role, seemingly led to neuroprotection by IGF-I.

Fungal Iron Availability during Deep Seated Candidiasis Is Defined by a Complex Interplay Involving Systemic and Local Events

Nutritional immunity – the withholding of nutrients by the host – has long been recognised as an important factor that shapes bacterial-host interactions. However, the dynamics of nutrient availability within local host niches during fungal infection are poorly defined. We have combined laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS), MALDI imaging and immunohistochemistry with microtranscriptomics to examine iron homeostasis in the host and pathogen in the murine model of systemic candidiasis. Dramatic changes in the renal iron landscape occur during disease progression. The infection perturbs global iron homeostasis in the host leading to iron accumulation in the renal medulla. Paradoxically, this is accompanied by nutritional immunity in the renal cortex as iron exclusion zones emerge locally around fungal lesions. These exclusion zones correlate with immune infiltrates and haem oxygenase 1-expressing host cells. This local nutritional immunity decreases iron availability, leading to a switch in iron acquisition mechanisms within mature fungal lesions, as revealed by laser capture microdissection and qRT-PCR analyses. Therefore, a complex interplay of systemic and local events influences iron homeostasis and pathogen-host dynamics during disease progression.

Analysis of innate defences against Plasmodium falciparum in immunodeficient mice

BackgroundMice with genetic deficiencies in adaptive immunity are used for the grafting of human cells or pathogens, to study human diseases, however, the innate immune responses to xenografts in these mice has received little attention. Using the NOD/SCID Plasmodium falciparum mouse model an analysis of innate defences responsible for the substantial control of P. falciparum which remains in such mice, was performed.MethodsNOD/SCID mice undergoing an immunomodulatory protocol that includes, clodronate-loaded liposomes to deplete macrophages and an anti-polymorphonuclear leukocytes antibody, were grafted with human red blood cells and P. falciparum. The systematic and kinetic analysis of the remaining innate immune responses included the number and phenotype of peripheral blood leukocytes as well as inflammatory cytokines/chemokines released in periphery. The innate responses towards the murine parasite Plasmodium yoelii were used as a control.ResultsResults show that 1) P. falciparum induces a strong inflammation characterized by an increase in circulating leukocytes and the release of inflammatory cytokines; 2) in contrast, the rodent parasite P. yoelii, induces a far more moderate inflammation; 3) human red blood cells and the anti-inflammatory agents employed induce low-grade inflammation; and 4) macrophages seem to bear the most critical function in controlling P. falciparum survival in those mice, whereas polymorphonuclear and NK cells have only a minor role.ConclusionsDespite the use of an immunomodulatory treatment, immunodeficient NOD/SCID mice are still able to mount substantial innate responses that seem to be correlated with parasite clearance. Those results bring new insights on the ability of innate immunity from immunodeficient mice to control xenografts of cells of human origin and human pathogens.

A Serotype 3 Pneumococcal Capsular Polysaccharide-Specific Monoclonal Antibody Requires Fcγ Receptor III and Macrophages To Mediate Protection against Pneumococcal Pneumonia in Mice

ABSTRACT Antibodies to pneumococcal capsular polysaccharide (PPS) are required for PPS-based vaccine-mediated protection against Streptococcus pneumoniae. Previous work established that 1E2, a mouse IgG1 to PPS3 that does not induce serotype 3 (ST3) S. pneumoniae killing by phagocytes in vitro, protects mice from death after intranasal infection with ST3, but its efficacy was abrogated in FcγR (F common gamma receptor)-deficient mice. In this study, we determined whether 1E2 efficacy against pulmonary ST3 infection requires FcγRIII. 1E2 did not protect FcγRIII-deficient (FcγRIII−/−) mice. Studies of the mechanism of 1E2-mediated effects showed that it resulted in a marked reduction in lung inflammation in ST3-infected wild-type (Wt [C57BL/6]) mice that was abrogated in FcγRIII−/− mice. 1E2 had no effect on early bacterial clearance in the lungs of ST3-infected Wt, FcγRIIB−/−, or FcγRIII−/− mice, but it reduced levels of bacteremia and serum macrophage inflammatory protein-2) (MIP-2), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) in Wt and FcγRIIB−/− mice, strains in which it is protective. As previous work showed that neutrophils were dispensable for 1E2 efficacy, we investigated whether macrophages are required for 1E2 efficacy against intranasal infection with ST3 and found that its efficacy was abrogated in Wt mice depleted of macrophages intranasally. In vitro studies revealed that1E2 promoted ST3 internalization by naïve alveolar macrophages but did not induce early intracellular killing. Macrophages from 1E2-treated ST3-infected mice studied ex vivo exhibited more apoptosis than those from FcγRIII−/− mice. These findings suggest that 1E2 mediates protection against ST3 in mice by affecting the inflammatory response, perhaps in part via macrophage apoptosis, rather than by inducing early bacterial clearance.

Myofiber Damage Precedes Macrophage Infiltration after in Vivo Injury in Dysferlin-Deficient A/J Mouse Skeletal Muscle

Mutations in the dysferlin gene (DYSF) lead to human muscular dystrophies known as dysferlinopathies. The dysferlin-deficient A/J mouse develops a mild myopathy after 6 months of age, and when younger models the subclinical phase of the human disease. We subjected the tibialis anterior muscle of 3- to 4-month-old A/J mice to in vivo large-strain injury (LSI) from lengthening contractions and studied the progression of torque loss, myofiber damage, and inflammation afterward. We report that myofiber damage in A/J mice occurs before inflammatory cell infiltration. Peak edema and inflammation, monitored by magnetic resonance imaging and by immunofluorescence labeling of neutrophils and macrophages, respectively, develop 24 to 72 hours after LSI, well after the appearance of damaged myofibers. Cytokine profiles 72 hours after injury are consistent with extensive macrophage infiltration. Dysferlin-sufficient A/WySnJ mice show much less myofiber damage and inflammation and lesser cytokine levels after LSI than do A/J mice. Partial suppression of macrophage infiltration by systemic administration of clodronate-incorporated liposomes fails to suppress LSI-induced damage or to accelerate torque recovery in A/J mice. The findings from our studies suggest that, although macrophage infiltration is prominent in dysferlin-deficient A/J muscle after LSI, it is the consequence and not the cause of progressive myofiber damage.