Neurons exhibit Lyz2 promoter activity in vivo: Implications for using LysM‐Cre mice in myeloid cell research

To characterize LysM-Cre mediated gene targeting in mice, we crossed LysM-Cre mice to two independent reporter-mouse lines (tdTomato or YFP). Surprisingly, we found that more than 90% of cells with LysM-Cre mediated recombination in the brain were neurons, rather than myeloid cells, such as microglia. Hence, by using the LysM-Cre mouse line for conditional knockout approaches, a significant neuronal recombination needs to be considered. Lysozymes are enzymes of the innate immune system found in plants, animals, bacteria, and fungi that cleave peptidoglycanes of bacterial cell walls. In humans, lysozyme is encoded by one gene, while mice have two distinct genes, Lyz1 and Lyz2. Lyz1-mediated lysozyme expression is restricted to Paneth granule cells in the intestines 1-3, while lysozyme encoded by the Lyz2 gene, called Lysozyme M (LysM), is thought to be expressed in myeloblasts, immature and mature macrophages, and neutrophils 2. Since its discovery, lysozyme has been used as a cell-specific marker for myeloid cells. Accordingly, Clausen et al. 4 generated a myeloid-specific Cre-recombinase (Cre)-expressing mouse line by inserting Cre-cDNA into the translational start site of the endogenous LysM gene. In combination with two different loxP-mouse lines, they showed a specific and highly efficient deletion of loxP-site flanked target genes in myeloid cells, but not in B or T cells. Faust et al. 5 adapted this strategy to create a LysM-enhanced green fluorescent protein (EGFP) reporter-mouse model and confirmed EGFP expression in most myelomonocytic cells. In addition, LysM-Cre mice were used to perform fate mapping analyses of LysM-Cre positive cells 6. LysM activity was shown not only in myeloid cells, but also in a subset of lymphoid and erythroid cells derived from LysM-Cre positive cells 6, suggesting that lysozyme is also expressed in early multipotent progenitor cells at some point during development. Furthermore, LysM-Cre mice have been used to genetically target myeloid cells or microglia (Supporting Information refs. 6–10). However, recently LysM-Cre mice have been controversially discussed to target microglia 7, 8. Therefore, we aimed at characterizing LysM-Cre positive cells by breeding LysM-Cre mice to tdTomato reporter mice 9 resulting in bright tdTomato expression in cells that either currently express Cre, driven by the endogenous LysM promoter, or have arisen from LysM-expressing cells. Either way, tdTomato expression labels cells that would be targeted by conditional knockout approaches using LysM-Cre mediated excision of target genes. In addition to the established LysM expression pattern in peripheral organs (Supporting Information Fig. 1), we found a robust neuronal expression of tdTomato induced by the LysM promoter in a subset of neurons (Fig. 1 and 2), indicating LysM-promoter activity in neurons or neuronal progenitor cells. Surprisingly, in some brain regions the vast majority of tdTomato-expressing cells could be characterized as neurons. Importantly, in tdTomato mice in the absence of Cre, no cells expressing tdTomato could be detected (Supporting Information Fig. 2A and B). To further verify LysM-driven recombination in brain neurons, we crossed LysM-Cre mice to yellow flourescent protein (YFP) reporter mice as a second reporter-mouse line, resulting in equal neuronal expression of the reporter protein as seen in tdTomato mice (Supporting Information Fig. 2C–E). In the neuronal layer of the motorcortex or the granule cell layer of the cerebellum 93.4 (±3.5)% and 98.7 (±1.5)%, respectively, of all tdTomato-positive cells were neurons, rather than microglia (n = 3). Activity of the LysM promoter in cortical neurons or their precursor cells could also be shown by bright tdTomato expression of commissural and projection fibers of these neurons in the corpus callosum (Fig. 2A and B) and internal capsule (Fig. 2A and C). Quantification of an average of 1500 NeuN-stained neurons per region and mouse substantiated differences between different brain regions. In the motorcortex (Fig. 1C and E and Supporting Information Fig. 4) ∼10% of all neurons expressed tdTomato, whereas ∼20% of granule cells in the cerebellum (Fig. 1D and E and Supporting Information Fig. 5) exhibited tdTomato expression. Additionally, tdTomato-positive Purkinje cells could be detected (Supporting Information Fig. 5). Also within the hippocampus formation, regional differences regarding the LysM-Cre driven tdTomato expression were detected. In the cornu ammonis region 3 (CA3), ∼10% of neurons were tdTomato-positive. In sharp contrast, tdTomato expression was almost completely absent in neurons of the gyrus dentatus or CA1 and CA2 (Fig. 1B and E and Supporting Information Fig. 6). We could confirm a low LysM-Cre mediated tdTomato expression by microglia with brain region-specific differences. In the hippocampus (Fig. 1A, D, and E) as well as the cerebellum (Fig. 1A, D, and E) an average of 18 and 27%, respectively, of Iba1-positive microglia expressed tdTomato, whereas only 5% of microglia in the motorcortex exhibited tdTomato expression (Fig. 1A, C, and E). Since fixation could impact on detection of fluorescent proteins, we confirmed our data using flow cytometry of acutely isolated living microglia of the entire forebrain defined by CD45 intermediate expression as described previously 10. In accordance with image quantification data, the average of tdTomato-positive microglia was 10.7% (n = 4; Supporting Information Fig. 3). Similar quantitative differences of LysM-positive microglia have been reported recently 11. Furthermore, LysM-Cre driven overexpression of methyl CpG binding protein 2 (MeCP2) was earlier used to implicate microglia in pathogenesis of the Rett syndrome, an autism spectrum disorder 12. These findings were challenged by a different study showing that MeCP2 expression is not restricted to microglia using the LysM-Cre mouse line. In line with our results, the authors also report a LysM-Cre dependent overexpression of MeCP2 in neurons 11. Hence, we extend the concerns regarding the efficient targeting of microglia using LysM-Cre mice 7, 8 by showing substantial neuronal DNA recombination. As expected, in all peripheral organs we studied (spleen, adipose tissue, liver, and lung) almost all peripheral macrophages, identified by Iba1 staining, exhibited bright tdTomato fluorescence (Supporting Information Fig. 1A–F). Importantly, in the muscular layer of the intestines also tdTomato-positive nerve fibers were found (Supporting Information Fig. 1G and H), suggesting that subpopulations of peripheral neurons also exhibit LysM-promoter activity. However, in the lung there was an additional tdTomato-positive cell population, which could not be co-stained by the macrophage marker Iba1. These cells were located in the alveolar walls and exhibited a spheroid morphology, characteristics of alveolar type II cells. Indeed, LysM-promoter activity has been described previously in alveolar type II cells 5. As outlined above, tdTomato expression could also be caused by irreversible tdTomato expression in neurons due to LysM-Cre driven recombination in neuronal progenitor cells during development, rather than current LysM-promoter activity. Of note, neurons and astrocytes share common precursor cells 13, and in this scenario astrocytes could also express tdTomato. In our study, we never detected tdTomato-positive cells with astrocyte morphology and glial fibrillary acid protein stainings revealed no tdTomato expression in glial fibrillary acid protein-positive astrocytes (Supporting Information Fig. 2F–H), suggesting that LysM promoter is not active in neural precursor cells, but is activated in neurons when these cells are already restricted to the neuronal lineage. Furthermore, we investigated whether the number of tdTomato-positive neurons could be increased, as this would indeed implicate a potential activity of the LysM promoter in mature neurons. To verify this hypothesis, we used an organotypic slice culture model of the hippocampus. In this model, preparation of hippocampal brain slices leads to an artificial activation of immune cells and neurons due to partial axotomy of neuronal projections 14. Indeed, we observed an increased number of tdTomato-positive microglia on days 3 and 7 ex vivo, compared to directly fixed slices at day 0 (Fig. 2D–I). More importantly, also the number of tdTomato-positive neurons increased within 7 days of cultivation, especially in the entorhinal cortex (Fig. 2D–I). Hence, in neurons the LysM promoter is regulated and can be activated by environmental cues. Nevertheless an immunohistochemical detection of lysozyme, the gene product of the Lyz2 locus, could not be achieved by antibody staining. In contrast, lysozyme staining of the intestines resulted in an intense staining of Paneth granule cells (data not shown), indicating no active lysozyme production of brain neurons under healthy conditions. The functional relevance of LysM expression in neurons is unknown. Lysozyme was reported to be upregulated in meningitis and after brain trauma, and it is thought to play a role in neurodegenerative diseases as a part of amyloid plaques 15-17. In these studies, lysozyme secretion was attributed to invading granulocytes or microglia. Our findings in mind, a neuronal source of lysozyme might be possible. In support of this hypothesis, Ohmi et al. 18 found a sevenfold increase in LysM-gene expression in neurons of the medial entorhinal cortex in a mouse model of Sanphillipo syndrome, a lysosomal storage disease with intraneuronal accumulation of several molecules, including lysozyme. In their study, the authors reported a profound accumulation of intracellular lysozyme in neurons as well as hyperphosphorylated tau (p-tau) in the same neurons and an age-dependent increase of p-tau in the gyrus dentatus. Since intracerebral application of lysozyme induced tau-phosphorylation in rats 19, a functional relation of lysozyme has been suspected in neurodegenerative diseases. In line with this hypothesis, LysM has been shown to be upregulated in a mouse model of Alzheimer's disease 20. Despite the putative function of lysozyme in neurons, we here report that approximately 90% or almost 100% of the LysM-promoter activity in the forebrain (motorcortex) and cerebellum (granule cell layer), respectively, is restricted to neurons, and not to microglia. These findings should be considered when using LysM-Cre mice to target myeloid cells, especially in the field of neuroimmunology. However, despite the low expression of LysM in microglia, as reported previously, LysM-Cre mice are still widely used to genetically alter expression of target genes in microglia. In 2014 and 2015 at least six publications used LysM-Cre mice and analyzed effects on the central nervous system (Supporting Information refs. 10–16). We here show that putative myeloid targeting using LysM-Cre mice would also affect a large subset of neurons, which may even be more pronounced under pathological conditions. Hence, by using the LysM-Cre mouse line to influence myeloid-specific target genes a significant neuronal recombination needs to be considered. We thank Martin Krüger for helpful discussions and Kathrin Jäger and Andreas Lösche from the FACS core unit (Leipzig University). This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG-SFB 1052/1: “Obesity mechanisms,” Project A04 to I.B. and J.E.), DFG-FOR1336 (Project B2 to I.B.), and by the Helmholtz alliance “Imaging and Curing Environmental Metabolic Disease” (I.B.). The authors declare no financial or commercial conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Figure 1. TdTomato expression in peripheral tissues. Cryosections from spleen (A), perigonadal adipose tissue (C), lung (D), intestine (E) and liver (F) of adult Lys-Tomato mice stained against the macrophage marker Iba1 (green) and the nuclear counterstain DAPI (blue) are presented. (B) Spleen cryosections of control mice did not show tdTomato expression (red; right side). (G and H) Higher magnification of the intestinal muscular layer stained against the glia cell marker GFAP (G) or the neuronal marker Calretinin (H). Arrows mark tdTomato-positive fibers. Images are representative of 3 independent experiments. Figure 2. Control experiments exclude tdTomato leakiness in control mice or tdTomato expression in astrocytes and verify neuronal lineage activity of LysM using YFP reporter mice. Figure 3. Analysis of tdTomato expression in living microglia by flow cytometry Figure 4. TdTomato expression in neurons in the motorcortex. Figure 5. TdTomato expression in neurons in the cerebellum and brainstem Figure 6. Region-specific tdTomato expression in neurons in the hippocampus Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.