Shown in figure are the heatmaps of differentially expressed genes in the lungs of A/J and B6 mice exposed to GMA-MS, GMA-SS welding fume or vehicle at 4 (panel A) and 16 weeks (panel B) post-exposure. Comparisons were made to the corresponding control mouse strain. Overall, at 4 and 16 weeks, expression patterns of the analyzed genes were more similar within exposure groups rather than between exposure groups. This indicates that a good consistency across samples was found on the individual arrays. Using our statistical criteria, 36 annotated genes resulted in 5 distinct subclusters among the A/J and B6 welding fume-exposed and control groups at 4 weeks post-exposure. The subclusters intermixed in the GMA-MS or -SS exposed B6 representing similar gene expression patterns within this strain to these welding fumes; this was in contrast to the A/J strain (see panel A).
Figure 1 Hierarchical clustering of differentially expressed genes in GMA welding fume-exposed A/J and B6 mice. Hierarchical clustering analysis of differentially expressed genes in the lungs of A/J and B6 mice exposed to GMA-MS or GMA-SS welding fume or PBS (sham) (more ...)
By 16 weeks post-exposure, 35 annotated genes resulted in 5 distinct subclusters among the A/J and B6 welding fume-exposed and control groups. In contrast to our 4 week analysis, the subclusters intermixed in the GMA-MS or -SS exposed A/J, representing similar gene expression patterns within this strain to these welding fumes by 16 weeks (see panel B).
Gene activation 4 and 16 weeks post-exposure to welding fume
Based on our selected analysis criteria, at 4 weeks after GMA-MS fume exposure the A/J strain had an overall upregulation in gene transcription compared with the B6. Nearly three quarters (32 out of 43) of the genes in the A/J lung were upregulated versus only 40% (8 out of 20) in the B6 strain at this time point (Figure , panel A). By 16 weeks post-exposure, the A/J exhibited an overall downregulation in gene transcription after GMA-MS compared with the B6, 69% (22 out of 32) versus 50% (9 out of 18), respectively (Figure , panel B). Similarly, with GMA-SS exposure, 88% (43 out of 49) of the genes were upregulated in the A/J, whereas 45% (10 out of 22) in the B6 were upregulated (Figure , panel A). At 16 weeks post-exposure to GMA-SS, the number of differentially expressed genes in the A/J was 35 versus 12 in the B6 strain. Of the genes analyzed, 83% (10 out of 12) in the B6 and 57% (20 out of 35) in the A/J strain were upregulated (Figure , panel B). These data collectively show a more marked response in the A/J at both time points and with both welding fumes.
Figure 2 Differential gene regulation after GMA-MS welding fume exposure in A/J and B6 mice. Comparison of the number of differentially expressed genes in the lungs of A/J and B6 mice exposed to GMA-MS welding fume at 4 (panel A) and 16 weeks (panel B) post-exposure. (more ...)
Figure 3 Differential gene regulation after GMA-SS welding fume exposure in A/J and B6 mice. Comparison of the number of differentially expressed genes in the lungs of A/J and B6 mice exposed to GMA-SS welding fume at 4 (panel A) and 16 weeks (panel B) post-exposure. (more ...)
4 weeks post-exposure to GMA-MS: IPA analysis
IPA analysis is unbiased and independent of the study design. The networks generated from the input of transcriptional data yields networks based on the known functions and interconnectivity of the affected genes. Therefore, network titles refer to the primary functions of the gene pathways. Network analysis shows upregulated (intensity of red) and downregulated (intensity of green) molecules with the remaining pathway molecules incorporated by IPA. Molecules that were not user specified, but incorporated into the network through relationships with other molecules are white and those that were neither up nor down regulated or did not meet the defined cutoff criteria are gray. The top network in the A/J lung at 4 weeks post-exposure to GMA-MS was behavior, nervous system development and function and gene expression which incorporated 19 focus genes out of 43 network eligible genes (Figure , panel A). Genes commonly associated with an inflammatory lung response were altered including kruppel-like factor 2 (lung) [KLF2
], chemokine (C-C motif) ligand 2 (CCL2
), chemokine (C-C motif) receptor 8 (CCR8)
and nuclear factor interleukin 3 regulated (NFIL3
). Predicted involvement, by IPA, of other molecules involved in this network were the nuclear factor-kappa B (NFκB) complex, platelet-derived growth factor BB (PDGF BB), p38 mitogen- activated protein kinase (MAPK) and the phosphoinositide 3-kinase (PI3K) complex. Lending to the title of this network was the alteration of genes under the higher level function of behavior and nervous system development and function. These genes including D site of albumin promoter (albumin D-box) binding protein (DBP
), period circadian protein homolog 2 (PER2
), and nuclear receptor subfamily 1, group D member 1 and 2 (NR1D1
are important in circadian rhythm signaling, but also may have functional roles in lung pathobiology and/or lung tumorigenesis [21
Figure 4 Top molecular networks 4 weeks after GMA-MS welding fume exposure in A/J and B6 mice. Gene network analysis by IPA of differentially expressed focus genes 4 weeks after exposure to GMA-MS welding fume in A/J (panel A) and B6 (panel B) mice. Whole datasets (more ...)
The response in the B6 GMA-MS-exposed lung involved a significant transcriptional downregulation and included genes involved in the higher level disease and disorder category of cancer, functional subcategory apoptosis (Figure , panel B). These genes included transcriptional regulators early growth response protein 1 (EGR1), KLF2, KLF4, nuclear receptor subfamily 4, group A, member 2 (NR4A2) and members of the v-fos FBJ murine osteosarcoma viral oncogene homolog family or FOS genes. An important macrophage-derived gene, interleukin 1 beta (IL1β), which regulates the acute phase response, was also downregulated at this time point.
16 weeks post-exposure to GMA-MS: IPA analysis
At the later time point after GMA-MS exposure in the A/J, predicted involvement of oncogenes associated with cell survival pathways included tumor protein 53 (TP53) that encodes p53 protein and v-myc myelocytomatosis viral oncogene homolog [avian] (MYC) (Figure , panel A). Genes both up and downstream from TP53 and MYC were those with functional roles in cellular stress responses and/or cell death and included DnaJ homolog subfamily B member 1(DNAJB1), heat shock protein 105 kDa (HSPH1) and zinc finger and BTB domain containing 16 (ZBTB16) which was also upregulated at 4 weeks (2.4 fold). Interestingly, continued involvement of circadian rhythm signaling genes (second highest rated network) was also found. At 16 weeks, predicted molecules included the NFκB family of transcription factors, particularly v-rel reticuloendotheliosis viral oncogene homolog A (avian) or RELA.
Figure 5 Top molecular networks 16 weeks after GMA-MS welding fume exposure in A/J and B6 mice. Gene network analysis by IPA of differentially expressed focus genes 16 weeks after exposure to GMA-MS welding fume in A/J (panel A) and B6 (panel B) mice. Whole datasets (more ...)
The response in the B6 GMA-MS-exposed lung at 16 weeks involved 8 genes that were upregulated in the top network including the inflammatory cytokines CCL2 and chemokine (C-X-C motif) ligand 2 (CXCL2) (Figure , panel B). A behavioral gene subset was differentially regulated in the B6 at 16 weeks and this network component was also present in the top network of the A/J strain at 4 weeks post-exposure to GMA-MS fume. A conserved, consistent expression of one of the behavioral genes NR1D1 was found. Expression levels were 2.3 and 2.2 fold for NR1D1 at 4 and 16 weeks, respectively. NFκB was a predicted molecule at this time point which formed a direct relationship with interleukin 1 (IL-1)-induced inflammatory gene, LCN2, or oncogene 24p3.
Summary of network discovery after GMA-MS welding fume exposure
In our previous study, at 4 weeks after GMA-MS welding fume exposure, minimal but significant lung cytotoxicity and inflammation persisted in the A/J strain, whereas inflammation resolved in the B6 by 7 days [9
]. Our lung transcriptome profiling in these mouse strains complements these findings. More specifically, an attenuated downregulation of the transcriptome and a greater number of affected genes in the A/J strain compared to the B6 was found. Some gene networks altered during the early and late resolution phases of the lung response to GMA-MS fume were not as expected. Although, anti-inflammatory signaling was occurring in the B6 at 4 weeks (i.e., downregulation of IL1β
, S100 calcium binding protein A9 [S100A9
], etc.) other "later" gene interactions were surprising (Figures and ). Perhaps the most intriguing finding regarding GMA-MS welding fume exposure was the differential expression of behavioral genes associated with circadian rhythm signaling. Most notably, we found a consistent increased expression of NR1D1
in both mouse strains at 4 and 16 weeks post-exposure. Although primarily characterized as a circadian rhythm regulatory gene, NR1D1
is implicated as a tumor suppressor gene and may modulate cell proliferation/differentiation and NF-κB pathways, a common hub in the GMA-MS gene networks [23
]. Consistent with our findings, previous studies also revealed changes in murine lung expression of circadian rhythm genes, including NR1D1
, following cigarette smoke exposure [21
]. These findings suggest that this particular gene subset may be important in the lung response to toxic stimuli.
4 weeks post-exposure to GMA-SS: IPA analysis
In the A/J GMA-SS-exposed lung, the top network included 22 significantly upregulated focus molecules such as inflammatory chemokines regulating cell (monocyte, natural killer, and neutrophil) movement such as CCL2 and CCL4 and CXCL2 (Figure , panel A). Increased transcriptional activity was also found for genes involved in the higher level disease and disorder category of immunological disease including the acute phase response protein serum amyloid 2 (SAA2), ZBTB16 and osteopontin. Predicted molecular involvement in this network were the Akt protein family (protein kinase B), the NFκB complex, activator protein-1 (AP-1), p38 MAPK and Mek.
Figure 6 Top molecular networks 4 weeks after GMA-SS welding fume exposure in A/J and B6 mice. Gene network analysis by IPA of differentially expressed focus genes 4 weeks after exposure to GMA-SS welding fume in A/J (panel A) and B6 (panel B) mice. Whole datasets (more ...)
The top network in the B6 GMA-SS-exposed lung consisted primarily of decreased gene expression for dual specificity phosphatase 1 (DUSP1), a downregulator of MAPK signaling, transcriptional regulators EGR1, FOS, FOSB, and pro-inflammatory cytokine IL1β (Figure , panel B). These gene interactions were also present in the B6 response to GMA-MS welding fume, which suggests similar transcriptional regulation to both MS and SS fumes in this strain at 4 weeks (Figures and ). Cellular movement, a top molecular and cellular function associated with GMA-SS in the B6, encompassed an overall downregulation of a gene subset involved in movement of leukocytes, lymphatic system and blood cells; these included colony stimulating factor 3 receptor [granulocyte] (CSF3R), DUSP1, IL1β, MMP9, S100A8 and S100A9.
16 weeks post-exposure to GMA-SS: IPA analysis
In one of the top two A/J networks, immune response, cell morphology, hematological system development and function, primarily consisted of upregulated genes which were chemokines CCL3
, and CXCL9
as well as immunoglobulin M (IgM
(Figure , panel A). Transcription of macrophage metalloelastase or MMP12
was an important and sustained response to both GMA-MS and -SS welding fume in the A/J strain. In contrast, increased MMP12
was evident only in response to GMA-SS welding fume at 4 weeks in the B6 lung, but was the top upregulated gene (1.8 fold). Expression levels of this proteolytic gene are primarily associated with macrophages; therefore, its overexpression may reflect an ongoing macrophage accumulation and/or activation in the A/J lung. Further, MMP12
was recently shown to play a key role in welding fume-induced lung inflammation as well as in fibrotic diseases such as asbestosis [12
]. Nine genes in the other top network, drug metabolism, lipid metabolism and small molecule biochemistry were upregulated including cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1
), ubiquitin D (UBD
), cholesterol 25-hydroxylase (CH25H
), delta-like 1 homolog [Drosophila] (DLK1
), and interleukin 4 induced 1 (IL41
) (Figure , panel B). These genes all had indirect connectivity to the main predicted gene hub in this network, tumor necrosis factor [TNF superfamily, member 2] (TNF
Figure 7 Top molecular networks 16 weeks after GMA-SS welding fume exposure in A/J and B6 mice. Gene network analysis by IPA of differentially expressed focus genes 16 weeks after exposure to GMA-SS welding fume in A/J (panel A & B) and B6 (panel C) mice. (more ...)
At 16 weeks, in the B6 GMA-SS-exposed lung the top network was associated with a similar network of genes as 4 weeks post-exposure although the transcriptional activation switched from decreased to increased (Figure , panel C). The genes CSF3R
, S100A8, S100A9
and resistin like beta (RETNLB
), downregulated at 4 weeks, were transcriptionally activated at 16 weeks post-exposure to GMA-SS fume. Some of these genes suggest neutrophil (S100A8 and A9
, interleukin 8 receptor beta [IL8Rβ]
) and macrophage recruitment (CCL2
) and/or perhaps a tissue remodeling response through activation of MMP9
. Although, because MMP9
is predominantly neutrophil-associated and some evidence suggests it may activate or potentiate IL-8, an inflammatory role in this study cannot be excluded [28
]. At 16 weeks, the expression of the reported tumor suppressor gene EGR1
, remained decreased as was found at 4 weeks post-exposure. Predicted molecules included NFκB, which formed a direct connection with MMP9
, Akt, PI3K, several members of MAPK and metal-regulatory transcription factor 1 (MTF1
Summary of network discovery after GMA-SS welding fume exposure
Considering the results of our previous comparative study, the A/J strain was predicted to have a sustained lung transcriptional response to GMA-SS welding fume compared to the B6 [9
]. Indeed, this was confirmed by our results, which may further suggest that the A/J strain lacks a necessary anti-inflammatory component to efficiently resolve the lung response to GMA-SS fume. Even at 16 weeks post-exposure, this lung tumor susceptible strain displayed chronic activation of immune response gene networks that included chemokines (CCL3
, etc) and various immunomodulatory factors such as LCN2
(> 2.5 fold increased at both time points) and SAA2
. This chronic gene activation to GMA-SS fume supports our long-term histopathological evidence for the presence of perivascular/peribronchial associated lung lymphoid infiltrates--composed of lymphocytes, macrophages, and plasma cells--in the A/J lung at 78 weeks post-exposure and also provides a rationale for further investigation into an enhanced tumorigenic potential of this fume. In contrast, as expected, the B6 exhibited an overall transcriptional downregulation of chemotactic gene signaling, but the later switch to an overexpression for this gene network was surprising. Vast evidence is emerging for the involvement of the leukocyte chemotaxis genes S100A8 and
(calgranulins) in inflammation-associated cancer which makes the co-upregulation at 16 weeks in the B6 strain intriguing [29
]. The dysregulation of the calgranulins S100A8
, in addition to CCL2
, warrants further investigation into a possible delayed inflammatory, fibrotic or perhaps proliferative response in this lung tumor resistant strain. Furthermore, involvement of MMP9
and possibly of the "cell-survival" Akt signaling pathway in the B6 may represent a generalized lung response to carcinogenic metals. Mechanistic data in the BALB/cJ mouse, an intermediate lung tumor susceptible strain, exposed to repetitive particulate Cr (VI) suggests these genes are important in the lung genotoxic response to this metal [30
Functional analysis of the lung response after GMA-MS and GMA-SS exposure
Reported in tables , , and are the associated categories of diseases and disorders, molecular and cellular functions and physiological system development and functions for A/J and B6 mice 4 and 16 weeks post-exposure to GMA-MS and -SS welding fume. The range of associated p-values indicates that each higher level functional category contains more than one lower level functional category. Within each higher level category, the number and regulation of the genes is shown. As presumed, genes often overlapped among the functional categories within a strain. Some functional categories overlapped, but gene subsets were different between the strains as predicted from the network analysis.
Functional analysis for the lung response 4 weeks post-exposure to GMA-MS welding fumea
Functional analysis for the lung response 16 weeks post-exposure to GMA-MS welding fumea
Functional analysis for the lung response 4 weeks post-exposure to GMA-SS welding fumea
Functional analysis for the lung response 16 weeks post-exposure to GMA-SS welding fumea
The 4 week analysis revealed genes associated with cancer that functioned primarily in cell death were significant in B6 lung response to GMA-MS welding fume (Table ). These included, for example, the FOS gene family, KLF2, KLF4, EGR1 and IL1β, all downregulated, and upregulated genes angiopoietin-related protein 4 (ANGPTL4), CCL6 and NR1D1. By 16 weeks, the majority of these genes were not differentially expressed in the B6 lung and the cancer category then included upregulated chemotactic genes (CCL2, CXCL2) which reflected the cell movement molecular and cellular function (Table ). Overall, however, the dominant disease and disorder categories in the B6 at both time points were connective tissue disorders, inflammatory disease and immune response which also predominated at 4 weeks in the A/J strain. At 16 weeks in the A/J lung, cancer genes were implicated in the late response to GMA-MS welding fume and cellular compromise was a significant function. In addition, the cell death response (LCN2, DNAJB1, ZBTB16, etc.), perpetuated at this time point and gene number was maintained from 4 weeks (Tables and ). This may indicate a defective or inadequate apoptotic response in the A/J lung after GMA-MS exposure. In summary, although cancer genes were implicated in both strains to GMA-MS welding fume, their primary role was likely not one of lung tumorigenesis in this experimental scenario.
The functional analysis of GMA-SS welding fume exposure confirmed that genes involved in inflammatory disease, immunological disease, connective tissue disorders and skeletal and muscular disorder were significant in both A/J and B6 mice (Table and ). However, as previously mentioned, the gene networks, numbers and regulation contrasted between the strains. The diseases and disorders that differed between the strains in response to GMA-SS fume at 4 and 16 weeks post-exposure were cancer in the A/J strain and hematological disease in the B6. Hematological disease included leukocytosis, or an increase in white blood cells (primarily neutrophils), as a lower level category. This confirmed the network analysis which showed that genes involved in this process (MMP9, S100A8, S100A9, etc.) initially downregulated, were then activated in the B6 lung at 16 weeks post-exposure. The interpretation of our finding that there is a switch from a protective, anti-inflammatory, response to a pro-inflammatory response in the B6 lung is difficult, but particle persistence in the lung may play a role.
Cancer persisted at 16 weeks only in the A/J strain and contained a large number of upregulated genes such as C13ORF15, LRG1, LCN2, MMP12 and SAA2 (Table ). Furthermore, at 16 weeks cellular growth and proliferation was an important molecular and cellular function that occurred in the A/J lung and differential regulation of genes such as IL4I, NR1D1, IGM, ZBTB16, and KLF4 were noted. Cell morphology (modification, shape change, and conversion of cells) and effects on cell cycle were also significant gene functions in the A/J lung after GMA-SS fume exposure and it may be of importance that these functions were not represented after GMA-MS fume exposure. Overall, the functional analysis indicates that this welding fume has continued deleterious effects on the lung which may enhance its tumorigenic potential in the A/J strain.
Confirmation of microarray gene expression by RT-qPCR
Validation of gene expression for CFB, LCN2, MMP12 and SPP1 was done by RT-qPCR from 4 week A/J GMA-MS and -SS lung samples (Figure ). Microarray results showed CFB and SPP1 were both increased 1.39 fold after GMA-SS exposure only, a value near the selected fold change cutoff for IPA; the significant induction was confirmed by real time RT-PCR supporting the expression value chosen for IPA. Similar corresponding validation was found for LCN2 and MMP12 (Figure ). Of note, the real time RT-PCR fold induction values were higher compared to microarray adding further to the utilization of a 1.3 fold cutoff.
Figure 8 RT-qPCR confirmation of microarray gene expression changes in welding fume-exposed A/J mice. Confirmation of microarray gene expression by RT-qPCR for CFB, LCN2, MMP12 and SPP1 in whole lung tissue from A/J mice 4 weeks post-exposure to GMA-MS or GMA-SS (more ...)