The genome-wide transcriptome data presented here demonstrate genotype and tissue-type related gene expression patterns in 9 V/null and 4L mice that are analogues of human Gaucher disease. Interestingly, the percentage of functional categories containing dysregulated RNAs was similar across all 9 V/null or 4L tissues, except for the macrophage activation genes that showed ~2-fold enrichment in 9 V/null lung, the most extensively involved organ (Table ). Another identified group was that for immune response genes, which were altered only in 9 V/null lung. Among the 54 immune response genes, 47 overlapped with the macrophage group. Since histological analyses did not find obvious T-cell and B-cell infiltration in 9 V/null lung, these immune responses likely emanated from macrophages. Macrophage activation in 9 V/null lung was also supported by hierarchical clustering analysis in which the 117 genes in cluster 3 (Figure ) were exclusively expressed in 9 V/null, rather than in 4L lung; a third of these were macrophage activation genes (Figure , highlighted region). These results indicate significant involvement of macrophage activation genes in 9 V/null lung, which was concordant with the extensive macrophage infiltration in the lungs. Moreover, the complementary histopathological studies showed that the nature of macrophage activation was not uniform between tissues or within macrophages in a specific tissue. These data support the concept of a variety of dysregulated macrophages that are tissue and disease state dependent as a dynamic component of the Gaucher disease/glucosylceramide storage pathophysiology.
Engorged macrophages are a hallmark of Gaucher disease, and these cells were differentially activated in tissues of 9 V/null and 4L mice at the mRNA, cellular, and immunohistochemical levels. The progressively increasing sizes and numbers of macrophages in visceral organs of Gba1
variant mice, because of the lipid storage, was particularly evident in lungs of 9 V/null mice from 12 to 28 wk (Figure ). Immunohistochemistry with the antibodies to selected IFNγ-regulated pro-inflammatory and IL-4-regulated anti-inflammatory cytokines showed that these cytokines were expressed in lung macrophages, but not in lung epithelial cells, i.e., there were pro- and anti-inflammatory responses (or activation) of the lipid-laden macrophage. Also, such analyses showed significant expression of the IFNγ protein in lung macrophages of 9 V/null mice at 12 and 28 wk (Figure ), but lesser expression in liver. These expression patterns were concordant with the differential activation of macrophages in these tissues as was evident by both microarray and morphologic data. In addition, IFNγ and its regulated pathway genes were less aberrant in tissues of the 4L mice, a more attenuated model, as ascertained by RNA or protein analyses. These results implicate pro-inflammation as a primary pathophysiological mechanism in Gaucher disease, as well as the degree of alterations in this pathway in the severity of Gaucher disease involvement. The production and secretion of cytokines from storage macrophages can be the important factors for the extracellular matrix components and function as they influence the interaction of surrounding macrophages with phagocytotic or endocytotic ligands and propagate the pathophysiology [21
IFNγ is a potent activator of macrophages and induces the expression of >300 genes, including those in the inflammatory mediator/chemokine pathway [22
]. Also, activated macrophages can be a significant source of IFNγ [23
]. Importantly, IFNγ inhibits macrophage proliferation and protects them from apoptosis [26
], thereby prolonging their survival within inflammatory loci [27
]. Here, the RNA expression levels of IFNγ, IL-6, NOS2, and TNFα were not significantly increased (FDR = 0.01), but their protein levels were specifically and highly expressed in the large lung macrophages. This observation indicates that the expression of these proteins and RNAs could be missed in whole tissue homogenates in which there may be large dilutional effects, if expression is restricted to specific cell types that make up a small percentage of total cells, e.g., macrophages. Importantly, 12 other cytokines are in the IFNγ pathway and could be downstream modulated by this cytokine. For example, IFNγ can induce TNFα and NOS2, and has differential effects on several individual chemokine genes [8
] that provide for selective stimulus sensitivity in mediating restricted pattern of chemokine gene expression. Transcripts for CXCL1/12 and the CCL chemokines (CCL2, 3, 6, 9 or 19) can be enhanced by Th1-related inflammatory mediators including IFNγ, IL-6, TNFα, or LPS [28
] as well as modulating the effects of each other (Figure ). The outcomes of such interactions lead to a cascading cytokine pro-inflammatory dysregulation that propagates Gaucher disease. Indeed, TNFα is a major regulator of chemokine gene expression, e.g., CXCL2, CCL3 and IL-6 [30
]. The finding here that TNFα protein was up regulated (Figure ) implicates its downstream cytokine network during Gaucher disease progression. Also, cytokines, e.g., CCL3, act synergistically with other macrophage chemokines [38
] to maintain the pro-inflammatory reactions.
Pro-inflammatory cytokines play a critical role in macrophage/leukocyte recruitment and adhesion [39
] and they recruit new macrophages to involved tissues via this cytokine network. Under the stimulus of accumulating glucosylceramide and other glucolipids, such a positive feedback macrophage-cytokine-macrophage cycle can be envisioned to expand and promote progression and the recruitment of additional pro-inflammatory cytokines/mediators networks (Figure ). The progressive cascade is schematically shown in Figure in which numerous interacting cytokines and chemokines are progressively up-regulated during disease progression from 4 to 28 wk. IFNγ central to this cascading network with initial mRNA up-regulation of β- and α-chemokines, as well as Mrs1 (macrophage scavenger receptor 1), TNFα, and NOS2. Among these interacting factors is the pleiotropic cytokine IL-6, which is a systemic alarm for tissue damage [43
]. The β-chemokines, CCL2, CCL3, CCL6, CCL9, CCL19, and the α-chemokines, CXCL1, CXCL12, mediate pro-inflammatory effects in the various types of cells and also have synergetic effects on their targets [30
]. The macrophage scavenger receptor 1 (Msr1) has been implicated in many macrophage-associated physiological and pathological processes through endocytosis [47
]. NOS2 and arginase were up-regulated at the RNA and protein levels in the 9 V/null mice, and as has been observed in ex vivo
]. NOS2 produces nitric oxide from arginine and stimulates pro-inflammation [49
]. NOS2 production in macrophages up-regulates vascular endothelial growth factor (e.g. VEGF) production and activates angiogenic activity [52
]. In comparison, arginase2 is a negative angiogenic regulator that inhibits NOS2 activity [54
]. Thus, arginase2 and NOS2 alternative pathways in activated macrophages [24
] and their up-regulation in 9 V/null mice simultaneous pro- and anti-inflammatory networks are being activated in the Gaucher disease process.
Interrogation of the IL-4 mediated anti-inflammatory network highlights significantly differential expression of 11 anti-inflammatory genes, indicating that participation of the aam
IL-4 pathway that counteract expression of macrophage pro-inflammatory cytokines and induce molecules that facilitate tolerance, healing and expression of innate immunity receptors, e.g. the scavenger receptor, CD163 [7
]. The IL-4 pathway (Figure ) displays the IL-4 time course and interactions over the 4 to 28 wk period. After an initial lag period from 4 to 12 wk, a network of such Th2 response genes [57
] is up-regulated at the RNA and/or protein levels. This network includes the structurally and functionally related matrix metalloproteinases MMP9/12/19 that are endopeptidases important to remodeling processes [9
]. These MMPs are among the most highly-expressed genes in most 9 V/null tissues. The high level expression of MMPs correlated with chronic fibrotic processes in 9 V/null lung and liver (unpublished observation). MMP12 expression occurs in human alveolar macrophages [18
] and airway smooth muscle cells [62
], and in murine alveolar type II epithelial cells [63
] or primary lung fibroblasts [64
]. The immunohistochemistry showed very strong signals in the lipid-laden macrophages, and much weaker signals in other lung cells (Figure ). The extremely high level of MMP12 expression (37 to 56-fold in 9 V/null lung and 16 to 22-fold in 9 V/null liver) implicates the aam
in Gaucher disease progression.
s are also implicated in the disease progression in 9 V/null mice as evidenced by CD163 expression [10
] and, particularly, expression of the IL-1 receptor antagonist (IL-1rn). IL-1rn inhibits the activities of IL-1A and IL-1B, and modulates IL-1 related immune and inflammatory responses. IL-1rn is typically produced by aam
and regulated by IL-4 [66
]. The interactions of these anti-inflammatory cytokines are schematically shown in Figure and indicate an overall description of pro-/anti-inflammatory networks in Gba1
The global gene expression networks integrate the gene expression patterns observed in 9 V/null or 4L mice. In these networks, more than 1/3 of significantly expressed genes were connected through the cascade interactions of pro- and anti-inflammatory genes. Although the macrophage involvement is major histopathological finding, about 3% of the genome was significantly altered at a molecular level during the development of the disease process. The propagation of the disease clearly depends on a generalized pro- and anti-inflammatory disruption.
Macrophages display marked phenotype heterogeneity in vitro
and in vivo
, including the responsiveness to endogenous and exogenous stimuli [8
]. Such heterogeneity results in differential phagocytosis or endocytosis, intracellular signaling and gene activation or repression [21
]. Macrophage heterogeneity was observed in the 9 V/null and 4L models by their differential activation and tissue distribution. Large lipid-laden macrophages (CD68 and F4/80 positive) were mostly observed in the lung of 9 V/null mice. Quantitative immunohistochemistry showed only some cytokines/effectors (i.e., IL-6, NOS2, CCL2, CCL3, CCL9, IL-4, and MMP12) were present in ~50% of lung macrophages (Figure ). However, IFNγ and Arg2 were present in nearly all of such macrophages. The corresponding RNAs of the cytokine/effectors were also up regulated. The basis for this heterogeneous expression of cytokine proteins in macrophages is unknown, but may be due to the different origins, differentiation sates, or maturation of the macrophage populations.
The genetic background of mice can influence gene expression profiles. Interstrain variations (1-3%) of gene expression profiles have been shown in different brain regions of mouse inbred strains [69
]. For example, such variation can be observed in the differential susceptibility to a wide range of pathogens [73
]. Here, 9 V/null and 4L mice had mixed strain backgrounds from three in-bred mouse strains FVB and/or C57BL/6J-129Sv. To evaluate the potential effects of mouse strain background on the expression profiles of macrophage activation genes, the WT data sets from FVB and three inbred mixed strains were used in the analyses. The result showed >50% of significantly expressed macrophage activation genes were shared when either of the two background controls were used. There was 83% concordance in the INFγ- and IL-4- regulated pathway genes. In addition, comparative analyses were conducted with duplicate lung RNA chip data from WT adult mice of three different genetic backgrounds (FVB, C57BL/6J, or 129Sv). The combined WT data generated 790 significantly expressed genes in 9 V/null lung (data not shown). About 60% of significantly expressed macrophage genes were concordant between FVB only or strain-matched WT controls, including >90% of INFγ-/IL4-regulated pathway genes (data not shown). The results showed general agreement of the gene expression profiles from individual WT backgrounds with those from WT controls either pure FVB or strain-matched backgrounds. The results show that macrophage activation genes are a significant functional group in the propagation of Gaucher disease in several genetic backgrounds.