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Papers by Robert Harris

Acta neuropathologica communications, Dec 1, 2020

CSF1R-related leukoencephalopathy is an adult-onset leukoencephalopathy with axonal spheroids and... more CSF1R-related leukoencephalopathy is an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia caused by colony stimulating factor 1 receptor (CSF1R) gene mutations. The disease has a global distribution and currently has no cure. Individuals with CSF1R-related leukoencephalopathy variably present clinical symptoms including cognitive impairment, progressive neuropsychiatric and motor symptoms. CSF1R is predominantly expressed on microglia within the central nervous system (CNS), and thus CSF1R-related leukoencephalopathy is now classified as a CNS primary microgliopathy. This urgent unmet medical need could potentially be addressed by using microglia-based immunotherapies. With the rapid recent progress in the experimental microglial research field, the replacement of an empty microglial niche following microglial depletion through either conditional genetic approaches or pharmacological therapies (CSF1R inhibitors) is being studied. Furthermore, hematopoietic stem cell transplantation offers an emerging means of exchanging dysfunctional microglia with the aim of reducing brain lesions, relieving clinical symptoms and prolonging the life of patients with CSF1R-related leukoencephalopathy. This review article introduces recent advances in microglial biology and CSF1R-related leukoencephalopathy. Potential therapeutic strategies by replacing microglia in order to improve the quality of life of CSF1R-related leukoencephalopathy patients will be presented.

International Journal of Molecular Sciences, 2020

Microglia, predominant parenchymal resident macrophages in the central nervous system (CNS), are ... more Microglia, predominant parenchymal resident macrophages in the central nervous system (CNS), are crucial players in neurodevelopment and CNS homeostasis. In disease conditions, pro-inflammatory microglia predominate over their regulatory counterparts, and are thus a potential immunotherapeutic target. It has been well documented that microglia can be effectively depleted using both conditional genetic Cx3cr1Cre-diphtheria toxin receptor (DTR)/diphtheria toxin subunit A (DTA) animal models and pharmacological colony-stimulating factor 1 receptor (CSF1R) inhibitors. Recent advances using these approaches have expanded our knowledge of the multitude of tasks conducted by microglia in both homeostasis and diseases. Importantly, experimental microglial depletion has been proven to exert neuroprotective effects in an increasing number of disease models, mostly explained by reduced neuroinflammation. However, the comprehensive effects of additional targets such as circulating monocytes and...

Targeting myeloid cells, especially microglia, for the treatment of neuroinflammatory diseases su... more Targeting myeloid cells, especially microglia, for the treatment of neuroinflammatory diseases such as multiple sclerosis (MS), is underappreciated. Here, we screened a library of compounds and identified the topoisomerase 1 (TOP1) inhibitor camptothecin (CPT) as a promising drug candidate for microglial modulation. CPT and its FDA-approved analog topotecan (TPT) inhibited inflammatory responses in microglia and macrophages, and ameliorated neuroinflammation in mice. Transcriptomic analysis of sorted microglia revealed an altered transcriptional phenotype following TPT treatment, with Ikzf1 identified as a potential target. Importantly, TOP1 expression was found elevated in several neuroinflammatory conditions, including human MS brains. To achieve targeted delivery to myeloid cells we designed a nanosystem using DNA origami and loaded TPT into it (TopoGami). TopoGami also significantly suppressed the inflammatory response in microglia and mitigated disease progression in MS-like mi...

Journal of Neuroinflammation, 2021

There are inherent structural and functional differences in the central nervous systems (CNS) of ... more There are inherent structural and functional differences in the central nervous systems (CNS) of females and males. It has been gradually established that these sex-specific differences are due to a spectrum of genetic, epigenetic, and hormonal factors which actively contribute to the differential incidences, disease courses, and even outcomes of CNS diseases between sexes. Microglia, as principle resident macrophages in the CNS, play a crucial role in both CNS physiology and pathology. However, sex differences of microglia have been relatively unexplored until recently. Emerging data has convincingly demonstrated the existence of sex-dependent structural and functional differences of rodent microglia, consequently changing our current understanding of these versatile cells. In this review, we attempt to comprehensively outline the current advances revealing microglial sex differences in rodent and their potential implications for specific CNS diseases with a stark sex difference. A...

Cancer management and research, 2018

IL-17 expressed by Th17 cells play a crucial role in tissue inflammation by induction of proinfla... more IL-17 expressed by Th17 cells play a crucial role in tissue inflammation by induction of proinflammatory and neutrophil mobilizing cytokines, and IL-17 polymorphisms are associated with colorectal cancer (CRC). We investigated the expression of IL-17 and the association of IL-17 gene polymorphisms with CRC susceptibility in a Middle East population. The study included 117 diagnosed CRC patients and 100 age- and gender-matched healthy controls. IL-17A rs2275913 (G197A) and IL-17F rs763780 (T7488C) single nucleotide polymorphisms, mRNA, and protein levels of IL-17A were assessed. We observed significant association between rs2275913 in IL-17A and susceptibility to CRC ( = 0.016228). The AG and AA genotypes conferred 2-fold and 2.8-fold, respectively, higher risk of developing CRC compared with individuals having GG genotype. Stratification of the data based on gender and age revealed very strong association of CRC with IL17A rs2275913 only in males and "AG" genotype in patie...

Nature Communications, 2018

Circulating monocytes can compete for virtually any tissue macrophage niche and become long-lived... more Circulating monocytes can compete for virtually any tissue macrophage niche and become long-lived replacements that are phenotypically indistinguishable from their embryonic counterparts. As the factors regulating this process are incompletely understood, we studied niche competition in the brain by depleting microglia with >95% efficiency using Cx3cr1CreER/+R26DTA/+ mice and monitored long-term repopulation. Here we show that the microglial niche is repopulated within weeks by a combination of local proliferation of CX3CR1+F4/80lowClec12a– microglia and infiltration of CX3CR1+F4/80hiClec12a+ macrophages that arise directly from Ly6Chi monocytes. This colonization is independent of blood brain barrier breakdown, paralleled by vascular activation, and regulated by type I interferon. Ly6Chi monocytes upregulate microglia gene expression and adopt microglia DNA methylation signatures, but retain a distinct gene signature from proliferating microglia, displaying altered surface marke...

Glia, 2018

Microglia are prominent immune cells in the central nervous system (CNS) and are critical players... more Microglia are prominent immune cells in the central nervous system (CNS) and are critical players in both neurological development and homeostasis, and in neurological diseases when dysfunctional. Our previous understanding of the phenotypes and functions of microglia has been greatly extended by a dearth of recent investigations. Distinct genetically defined subsets of microglia are now recognized to perform their own independent functions in specific conditions. The molecular profiling of single microglial cells indicates extensively heterogeneous reactions in different neurological disorders, resulting in multiple potentials for crosstalk with other kinds of CNS cells such as astrocytes and neurons. In settings of neurological diseases it could thus be prudent to establish effective cell-based therapies by targeting entire microglial networks. Notably, activated microglial depletion through genetic targeting or pharmacological therapies within a suitable time window can stimulate replenishment of the CNS niche with new microglia. Additionally, enforced repopulation through provision of replacement cells also represents a potential means of exchanging dysfunctional with functional microglia. In each setting the newly repopulated microglia might have the potential to resolve ongoing neuroinflammation. In this review, we aim to summarize the most recent knowledge of microglia and to highlight microglial depletion and subsequent repopulation as a promising cell replacement therapy. Although glial cell replacement therapy is still in its infancy and future translational studies are still required, the approach is scientifically sound and provides new optimism for managing the neurotoxicity and neuroinflammation induced by activated microglia.

Nature immunology, 2018

The cytokine transforming growth factor-β (TGF-β) regulates the development and homeostasis of se... more The cytokine transforming growth factor-β (TGF-β) regulates the development and homeostasis of several tissue-resident macrophage populations, including microglia. TGF-β is not critical for microglia survival but is required for the maintenance of the microglia-specific homeostatic gene signature. Under defined host conditions, circulating monocytes can compete for the microglial niche and give rise to long-lived monocyte-derived macrophages residing in the central nervous system (CNS). Whether monocytes require TGF-β for colonization of the microglial niche and maintenance of CNS integrity is unknown. We found that abrogation of TGF-β signaling in CX3CR1 monocyte-derived macrophages led to rapid onset of a progressive and fatal demyelinating motor disease characterized by myelin-laden giant macrophages throughout the spinal cord. Tgfbr2-deficient macrophages were characterized by high expression of genes encoding proteins involved in antigen presentation, inflammation and phagocyto...

Frontiers in aging neuroscience, 2017

While bone marrow-derived Ly6C(hi) monocytes can infiltrate the central nervous system (CNS) they... more While bone marrow-derived Ly6C(hi) monocytes can infiltrate the central nervous system (CNS) they are developmentally and functionally distinct from resident microglia. Our understanding of the relative importance of these two populations in the distinct processes of pathogenesis and resolution of inflammation during neurodegenerative disorders was limited by a lack of tools to specifically manipulate each cell type. During recent years, the development of experimental cell-specific depletion models has enabled this issue to be addressed. Herein we compare and contrast the different depletion approaches that have been used, focusing on the respective functionalities of microglia and monocyte-derived macrophages in a range of neurodegenerative disease states, and discuss their prospects for immunotherapy.

Neuromolecular medicine, Jan 21, 2017

In this study, we investigated the uptake of malondialdehyde (MDA)-modified myelin oligodendrocyt... more In this study, we investigated the uptake of malondialdehyde (MDA)-modified myelin oligodendrocyte glycoprotein (MOG) in the context of lipid peroxidation and its implications in CNS autoimmunity. The use of custom-produced fluorescently labeled versions of MOG or MDA-modified MOG enabled us to study and quantify the uptake by different macrophage populations and to identify the responsible receptor, namely SRA. The SRA-mediated uptake of MDA-modified MOG is roughly tenfold more efficient compared to that of the native form. Notably, this uptake is most strongly associated with anti-inflammatory M2-type macrophages. MDA-modified MOG was demonstrated to be resistant to degradation by lysine-dependent proteases in vitro, but the overall digestion fragments appeared to be similar in cell lysates, although their relative abundance appeared to be altered as a result of faster uptake. Accordingly, MDA-modified MOG is processed for presentation by APCs, allowing maximized recall proliferat...

Molecular brain, Jun 19, 2017

Microglia are the principal resident immune cells in the central nervous system and are believed ... more Microglia are the principal resident immune cells in the central nervous system and are believed to be versatile players in both inflammatory and physiological contexts. On the one hand, in order to safeguard the microenvironment microglia can be rapidly activated by contact with microbial products or cell debris, thereby exerting the functions of innate immunity via phagocytosis and secretion of cytokines and chemokines. Conversely, microglia can also assist in brain development, synaptic plasticity and neural repair through the production of neurotrophic factors and clearance of myelin debris. It is now well accepted that the dysfunction of microglia and microglia-induced neuroinflammation are implicated in the occurrence and progression of many neurological diseases. Although the past decade has witnessed major progress in understanding of multi-tasking microglia, what remains largely enigmatic is the relative importance of microglia at different disease stages and how microglia ...

Oncotarget, Jan 26, 2016

The relative contribution of resident microglia and peripheral monocyte-derived macrophages in ne... more The relative contribution of resident microglia and peripheral monocyte-derived macrophages in neuroinflammation after cranial irradiation is not known. A single dose of 8 Gy was administered to postnatal day 10 (juvenile) or 90 (adult) CX3CR1GFP/+ CCR2RFP/+ mouse brains. Microglia accumulated in the subgranular zone of the hippocampal granule cell layer, where progenitor cell death was prominent. The peak was earlier (6 h vs. 24 h) but less pronounced in adult brains. The increase in juvenile, but not adult, brains was partly attributed to proliferation. Microglia numbers then decreased over time to 39% (juvenile) and 58% (adult) of controls 30 days after irradiation, largely as a result of cell death. CD68 was expressed in 90% of amoeboid microglia in juvenile hippocampi but only in 9% of adult ones. Isolated hippocampal microglia revealed reduced CD206 and increased IL1-beta expression after irradiation, more pronounced in juvenile brains. CCL2 and IL-1 beta increased after irrad...

Glia, Nov 1, 2016

Intracerebral levels of Transforming Growth Factor beta (TGFβ) rise rapidly during the onset of e... more Intracerebral levels of Transforming Growth Factor beta (TGFβ) rise rapidly during the onset of experimental autoimmune encephalomyelitis (EAE), a mouse model of Multiple Sclerosis (MS). We addressed the role of TGFβ responsiveness in EAE by targeting the TGFβ receptor in myeloid cells, determining that Tgfbr2 was specifically targeted in monocyte-derived dendritic cells (moDCs) but not in CNS resident microglia by using bone-marrow chimeric mice. TGFβ responsiveness in moDCs was necessary for the remission phase since LysM(Cre) Tgfbr2(fl/fl) mice developed a chronic form of EAE characterized by severe demyelination and extensive infiltration of activated moDCs in the CNS. Tgfbr2 deficiency resulted in increased moDC IL-12 secretion that skewed T cells to produce IFN-γ, which in turn enhanced the production of moDC-derived reactive oxygen species that promote oxidative damage and demyelination. We identified SNPs in the human NOX2 (CYBB) gene that associated with the severity of MS,...

The Journal of experimental medicine, Jul 25, 2016

Prolonged infections or adjuvant usage can trigger emergency granulopoiesis (EG), leading to dysr... more Prolonged infections or adjuvant usage can trigger emergency granulopoiesis (EG), leading to dysregulation in neutrophil blood counts. However, the impact of EG on T and B cell function remains largely unknown. In this study, to address this question, we used a mouse model of neutropenia and studied immune activation after adjuvant administration. The initial neutropenic state fostered an environment of increased dendritic cell activation and T cell-derived IL-17 production. Interestingly, neutropenic lysozyme 2-diphtheria toxin A mice exhibited striking EG and amplified neutrophil recruitment to the lymph nodes (LNs) that was dependent on IL-17-induced prostaglandin activity. The recruited neutrophils secreted a B cell-activating factor that highly accelerated plasma cell generation and antigen-specific antibody production. Reduction of neutrophil functions via granulocyte colony-stimulating factor neutralization significantly diminished plasma cell formation, directly linking EG w...

Oncoimmunology, 2016

In tumor patients, IL-6 appears to be one component of a consistent cancer-associated cytokine ne... more In tumor patients, IL-6 appears to be one component of a consistent cancer-associated cytokine network resulting in both a systemic immune stimulation and a microenvironment of cancer-induced immune suppression that ultimately protects the cancer cells. IL-6 has been associated with prognosis in cancer patients, but so far a systemical analysis has not been carried out. The present meta-analysis studies the relation between IL-6 serum levels and the prognosis of cancer patients in the available clinical literature of 100 articles published between 1993 and 2013 comprising 11,583 patients. The IL-6 serum level was described as significantly correlating with survival in 82/101 series comprising 85.6% of patients (9917/11,583) with 23 different cancer types. A total of 64 studies dichotomized patient cohorts according to various cut-off IL-6 serum levels: in 59/64 of these series corresponding to 94.5% of the reported patients (7694/8142) significant correlations between IL-6 serum lev...

Cell reports, May 19, 2016

Tumors are composed of multiple cell types besides the tumor cells themselves, including innate i... more Tumors are composed of multiple cell types besides the tumor cells themselves, including innate immune cells such as macrophages. Tumor-associated macrophages (TAMs) are a heterogeneous population of myeloid cells present in the tumor microenvironment (TME). Here, they contribute to immunosuppression, enabling the establishment and persistence of solid tumors as well as metastatic dissemination. We have found that the pattern recognition scavenger receptor MARCO defines a subtype of suppressive TAMs and is linked to clinical outcome. An anti-MARCO monoclonal antibody was developed, which induces anti-tumor activity in breast and colon carcinoma, as well as in melanoma models through reprogramming TAM populations to a pro-inflammatory phenotype and increasing tumor immunogenicity. This anti-tumor activity is dependent on the inhibitory Fc-receptor, FcγRIIB, and also enhances the efficacy of checkpoint therapy. These results demonstrate that immunotherapies using antibodies designed t...

Journal of proteome research, Jul 25, 2016

Innate immune cells are complex systems that can be simultaneously activated in a variety of ways... more Innate immune cells are complex systems that can be simultaneously activated in a variety of ways. Common methods currently used to estimate the response of innate immune cells to stimuli are usually biased towards a single mode of activation. The aim of this study was to assess the possibility of designing an assay based on unbiased proteome analysis that would be capable of predicting the complex response of the innate immune system to various challenges. Monocytes were used as representative cells of the innate immune system. The underlying hypothesis was that their proteome response to different activating molecules would reflect the immunogenicity of these molecules. To identify the main modes of response, the human monocytic THP-1 cell line was treated with nine different stimuli. Differentiation and activation were determined to be the two major modes of monocyte response with PMA causing the strongest differentiation and Pam3CSK4 causing the strongest proinflammatory activat...

Immunology and cell biology, Sep 14, 2016

While pro-inflammatory immune responses are a requirement to combat microbes, uncontrolled self-d... more While pro-inflammatory immune responses are a requirement to combat microbes, uncontrolled self-directed inflammatory immune responses are the hallmark of autoimmune diseases. Restoration of immunological tolerance involves both suppression of ongoing tissue-destructive immune responses and re-education of the host immune system. Both functionally immunosuppressive macrophages (M2) and regulatory T cells (Tregs) are implicated in these processes. Their mutual interaction is synergistic in this context and adoptive transfer of each cell type has been functioning as immunotherapy in experimental models, being particularly effective when using M2 macrophages generated with an optimized IL-4/IL-10/TGF-β combination. As a prerequisite for eventual translation of M2 therapy into clinical settings we herein studied the induction, stability and mechanism of generation of human induced Tregs (iTregs) by M2 macrophages generated with IL-4/IL-10/TGF-β. The supernatants of monocyte-derived huma...

Cancer research, Jan 15, 2016

Successful immunotherapy of cancer is becoming a reality aided by the realization that macrophage... more Successful immunotherapy of cancer is becoming a reality aided by the realization that macrophages play an important role in the growth or regression of tumors. Specifically, M2/repair-type macrophages predominate in human cancers and produce growth-promoting molecules that actively stimulate tumor growth in much the same way they help wounds heal. However, modulating M2/repair-type macrophages to M1/kill-type can slow or stop cancer growth. The effects involve direct activity of M1 kill-type as well as the ability of M1-type macrophages to stimulate Th1-type cytotoxic T cells and other effector cells. Macrophage responses can also predict cancer susceptibility; individuals with a high M1/kill to M2/repair ratio are less prone. That macrophages/innate immunity can be modulated to play a central role in directly or indirectly combating cancer is a breakthrough that seems likely to finally make successful immunotherapy of cancer a reality. Cancer Res; 76(3); 1-4. ©2016 AACR.

Journal of leukocyte biology, Jan 29, 2015

Dendritic cells are professional APCs that play a central role in the initiation of immune respon... more Dendritic cells are professional APCs that play a central role in the initiation of immune responses. The limited ex vivo availability of dendritic cells inspires the widespread use of bone marrow-derived dendritic cells as an alternative in research. However, the functional characteristics of bone marrow-derived dendritic cells are incompletely understood. Therefore, we compared functional and phenotypic characteristics of rat bone marrow-derived dendritic cells generated with GM-CSF/IL-4 or FLT3 ligand bone marrow-derived dendritic cells. A comparison of surface markers revealed that FLT3 ligand-bone marrow-derived dendritic cells expressed signal regulatory protein α, CD103, and CD4 and baseline levels of MHC class II, CD40, and CD86, which were highly up-regulated upon stimulation. Conversely, GM-CSF/IL-4-bone marrow-derived dendritic cells constitutively expressed signal regulatory protein α, CD11c, and CD11b but only mildly up-regulated MHC class II, CD40, or CD86 following st...