Quantitative Proteomic Analysis by Label Free
To investigate the changes in proteomic profiles of BAM, which was caused by the infection of MTB and MB, total proteins of normal BAM (control), MB- and MTB-infected BAM with three replicates were extracted, and then analyzed by LC/ESI-MS/MS and quantified by Label Free. Totally, 46018 spectra were generated, and 5467 proteins were identified against the bovine reference.
The two score plots of the PCA models show a clear separation of samples from different experimental groups (MTB and MB) and controls groups BAM, accounting for 36.6% of the observed variance, indicating that pathogenic bacteria infection was the key factor affecting protein expression (Fig. 1A). A genotype effect was also observed explaining about 21.6% of variance (Fig. 1A). Remarkably, the MB-infected samples showed a slight interaction with other two sample groups (MTB and BAM), suggesting that under MB infection challenge BAM could make some similar responses with MTB challenges (Fig. 1A). Additionally, correlation analysis displayed a well consensus with the PCA analysis that the variations of the three biological replicates were calculated according to their quantitative data, and they all showed little variation between three biological replicates, indicating a better quality and reproducibility of the data (Fig. 1B). Therefore, we proposed that MB-infection and MTB-infection will all result in significant changes in gene expression pattern of BAM, and some similar response could be invoked by both MB-infection and MTB infection. The same and different responses caused by both pathogenic bacteria will be the focus below.
Differentially expressed proteins in the bovine alveolar macrophages after being challenged by the MTB- and MB-infection
To determine the in-depth variation between MTB-infection and MB-infection, two pairwise comparisons were designed, including MTB.vs.BAM and MB.vs.BAM. In total, 18 proteins were significantly up or down-regulated during MTB infection (Fig. 2A, B; Log1.5FC < -1 or > 1, P < 0.05). We identified that 17 proteins were up-regulated and 1 protein was down-regulated during MTB infection (Fig. 2A). Only one host protein, regulator Prefoldin subunit 5 (Q8HYI9), was down-regulated by MTB infection. In parallel, 60 proteins were up-regulated and 3 proteins were down-regulated during MB infection (Fig. 2B). The three down-regulated proteins were TBC1 domain family member 2A, Polysaccharide biosynthesis domain containing 1 and Beta-defensin 10 (A6QP29, F1MV85 and P46168; Table 5).
In addition, to show the proteins which were up-regulated or down-regulated during MTB- and MB-infection BAM, up-set diagrams were shown as Fig. 2C. Among these up-regulated proteins, we further noted that 9 up-regulated proteins were shared by MTB- and MB-infection (Fig. 2C). Meanwhile, 51 proteins were only invoked to up-regulate by MB-infection and 8 proteins could be induced by MTB-infection (Fig. 2C). The results indicated that MB-infection could stimulate the BAM to make more responses than MTB-infection. However, no down-regulated proteins were shared by both MTB.vs.BAM and MB.vs.BAM (Fig. 2C). Based on this, three kinds of up-regulated proteins will be the analysis focus below to elucidate the similarities and differences in response of MTB- and MB-infected bovine cells.
Functional annotation based on GO and KEGG analysis on nine MTB- and MB-induced up-regulated proteins
In order to detect whether the nine up-regulated proteins were significantly enriched in certain functional types, Gene Ontology (GO) and KEGG enrichment analyses were performed on these proteins (Fig. 3; Table 2). To clearly investigate the similarities between MTB.vs.BAM and MB.vs.BAM pairwise comparisons, we firstly performed GO enrichment analysis on these nine up-regulated proteins (Fig. 3A, B, C; Table 2). We found that terms relevant to biological progresses were the most significant enriched categorizes, whereas terms involved in cellular component and molecular function were relatively less (Fig. 3A, B, C; Table 2). Being challenged by both MTB and MB pathogenic bacteria, BAM will activated various terms relevant to cellular component, including lysosomal lumen, lytic vacuole, lysosome and autophagosome (Fig. 3A; Table 2). It has been reported that lysosome-related proteins were involved in defense response of macrophage to MTB and MB infection [8, 9]. We further noted that many immunity-related terms which involved in molecular function were activated in response MTB- and MB-infection, including NADPH-hemoprotein reductase activity, interleukin-1 receptor binding, scavenger receptor activity, calcium-dependent protein binding and cytokine activity (Fig. 3B; Table 2). As being attacked by pathogenic bacteria, calcium-related signaling pathways will rapidly transmit the signal to activate the defense response, including a start of an inflammatory reaction[18, 19]. Importantly, in biological progress categorizes, many defense-related terms were significantly invoked by both pathogenic bacteria infection, including acute-phase response, cellular response to molecule of bacterial origin, cellular response to biotic stimulus, response to molecule of bacterial origin, acute inflammatory response, inflammatory response, defense response, response to bacterium, cellular response to oxygen-containing compound, regulation of antimicrobial humoral response, positive regulation of antimicrobial humoral response, response to biotic stimulus and regulation of inflammatory response (Fig. 3C; Table 2). Together, we concluded that the autophagy-related terms, inflammatory-related progresses and defenses to pathogenic bacteria were the important responses of BAM, which was invoked by the MTB and MB attack challenges.
To further investigate the role of up-regulated proteins in response to MTB and MB infection, these nine significantly up-regulated proteins from MTB- and MB-infected BAM were further annotated by KEGG pathway analysis with p < 0.05 against animal reference pathways from KEGG database (Fig. 3D). Up-regulated of Label Free quantitative proteins after infection of both pathogenic bacteria was mainly enriched in NF-kappa B signaling pathway, IL-17 signaling pathway, C-type lectin receptor signaling pathway, Tuberculosis, Cytokine-cytokine receptor interaction, Inflammatory mediator regulation of TRP channels, Toll-like receptor signaling pathway, Peroxisome and HIF-1 signaling pathway (Fig. 3D). These pathways mainly performed functions in inflammatory- and immunity-related responses of host cells as being attacked by pathogenic microbial. It has been proved that NF-kappa B signaling pathway, IL-17 signaling pathway and Toll-like receptor signaling pathway have important function in tuberculosis [10–12, 18, 19]. Therefore, we highlight that both MTB and MB infection could cause inflammatory responses of BAM.
Table 2
Significantly enriched Gene Ontology terms in both MTB- and MB-infected bovine alveolar macrophages
GO ID
|
Description
|
Gene Number
|
pvalue
|
Genes
|
GO:0043202
|
lysosomal lumen
|
1
|
0.010493
|
A0A3S5ZPN6
|
GO:0000323
|
lytic vacuole
|
2
|
0.030988
|
A0A3S5ZPN6; P09428
|
GO:0005764
|
lysosome
|
2
|
0.030988
|
A0A3S5ZPN6; P09428
|
GO:0005776
|
autophagosome
|
1
|
0.048991
|
P09428
|
GO:0003958
|
NADPH-hemoprotein reductase activity
|
1
|
0.009069
|
F1MYR5
|
GO:0005149
|
interleukin-1 receptor binding
|
1
|
0.012076
|
P09428
|
GO:0005044
|
scavenger receptor activity
|
1
|
0.018066
|
A0A3S5ZPN6
|
GO:0048306
|
calcium-dependent protein binding
|
1
|
0.026989
|
F1MX83
|
GO:0005125
|
cytokine activity
|
1
|
0.041702
|
P09428
|
GO:0006953
|
acute-phase response
|
2
|
1.04E-05
|
F1MNI5; P09428
|
GO:0071219
|
cellular response to molecule of bacterial origin
|
3
|
1.87E-05
|
F1MGW6; F1MYR5; P09428
|
GO:0071216
|
cellular response to biotic stimulus
|
3
|
3.27E-05
|
F1MGW6; F1MYR5; P09428
|
GO:0002237
|
response to molecule of bacterial origin
|
3
|
7.37E-05
|
F1MGW6; F1MYR5; P09428
|
GO:0002526
|
acute inflammatory response
|
2
|
9.64E-05
|
F1MNI5; P09428
|
GO:0006954
|
inflammatory response
|
3
|
0.0003
|
F1MNI5; F1MYR5; P09428
|
GO:0006952
|
defense response
|
4
|
0.000312
|
F1MGW6; F1MNI5; F1MYR5; P09428
|
GO:0009617
|
response to bacterium
|
3
|
0.000534
|
F1MGW6; F1MYR5; P09428
|
GO:1901701
|
cellular response to oxygen-containing compound
|
3
|
0.001038
|
F1MGW6; F1MYR5; P09428
|
GO:0002759
|
regulation of antimicrobial humoral response
|
1
|
0.002037
|
F1MGW6
|
GO:0002760
|
positive regulation of antimicrobial humoral response
|
1
|
0.002037
|
F1MGW6
|
GO:0009607
|
response to biotic stimulus
|
3
|
0.002496
|
F1MGW6; F1MYR5; P09428
|
GO:0050727
|
regulation of inflammatory response
|
2
|
0.00301
|
F1MNI5; F1MYR5
|
Functional analysis on up-regulated proteins which were specifically activated by MTB infection
Gene ontology analysis provides a generally accepted identification set to describe proteins attributes in an organism. Hence, we used GO functional annotation analysis on the up-regulated proteins which were only induced to over-express by MTB to elucidate the specific response of BAM under MTB challenges (Fig. 4A; Table 3). All these eight MTB-induced proteins were annotated with categories simultaneously. The classical three terms, molecular functions, biological processes and cellular components, were widely covered (Fig. 4A; Table 3). The results showed that the terms involved in molecular functions and cellular components were the main categorizes which invoked to significantly enriched in BAM by MTB infection (Fig. 4A; Table 3). For cellular component category, fourteen terms were significantly enriched, including fibrinogen complex, mitochondrial respiratory chain complex I, NADH dehydrogenase complex, respiratory chain complex I, mitochondrial respiratory chain, respiratory chain complex, respiratory chain, oxidoreductase complex, photoreceptor inner segment, inner mitochondrial membrane protein complex, mitochondrion, mitochondrial protein complex, mitochondrial part and mitochondrial membrane part (Fig. 4A; Table 3). The results further showed that all significantly enriched cellular component-related terms were involved in energy metabolism and performed functions in mitochondria, suggesting that the MTB infection will induce dramatic changes in energy metabolisms of BAM, which could help host cells to defense pathogenic bacteria attacks. Similarly, in molecular function category, the terms relevant to energy metabolisms were also the active terms of BAM in response to MTB infection, including NADH dehydrogenase activity, oxidoreductase activity, acting on NAD(P)H, lysophosphatidic acid phosphatase activity, oxidoreductase activity, acting on NAD(P)H, inorganic diphosphatase activity, aldehyde dehydrogenase (NADP+) activity and NADP-retinol dehydrogenase activity (Fig. 4A; Table 3). Therefore, we proposed that as being challenged by MTB infection, BAM will accelerate energy metabolism to defense the MTB attacks to protect the organisms.
The KEGG database was further analyzed to help determine the biological processes and functions of these eight MTB-activated proteins (Fig. 4B). All these eight proteins were mainly related to energy metabolism represented by Oxidative phosphorylation and immune system represented by Complement and coagulation cascades (Fig. 4B). Meanwhile, the significantly identified progress involved in response to pathogenic bacteria was Staphylococcus aureus infection (Fig. 4B). In conclusion, under MTB infection challenges, dramatic activation of energy metabolisms was the main motion which was performed in BAM in response to MTB attacks.
Table 3
Significantly enriched categories relevant to molecular function of eight MTB specifically activated proteins
GO ID
|
Description
|
Gene Number
|
pvalue
|
Genes
|
GO:0008137
|
NADH dehydrogenase (ubiquinone) activity
|
2
|
0.000887
|
P23935; P42028
|
GO:0050136
|
NADH dehydrogenase (quinone) activity
|
2
|
0.000887
|
P23935; P42028
|
GO:0003954
|
NADH dehydrogenase activity
|
2
|
0.00098
|
P23935; P42028
|
GO:0016655
|
oxidoreductase activity, acting on NAD(P)H
|
2
|
0.001076
|
P23935; P42028
|
GO:0052642
|
lysophosphatidic acid phosphatase activity
|
1
|
0.002358
|
A6H757
|
GO:0016651
|
oxidoreductase activity, acting on NAD(P)H
|
2
|
0.003218
|
P23935; P42028
|
GO:0004427
|
inorganic diphosphatase activity
|
1
|
0.004711
|
Q2KIV7
|
GO:0033721
|
aldehyde dehydrogenase (NADP+) activity
|
1
|
0.004711
|
E1BM93
|
GO:0052650
|
NADP-retinol dehydrogenase activity
|
1
|
0.004711
|
E1BM93
|
GO:0003993
|
acid phosphatase activity
|
1
|
0.007059
|
A6H757
|
GO:0004033
|
aldo-keto reductase (NADP) activity
|
1
|
0.009402
|
E1BM93
|
GO:0008106
|
alcohol dehydrogenase (NADP+) activity
|
1
|
0.009402
|
E1BM93
|
GO:0016491
|
oxidoreductase activity
|
3
|
0.017809
|
E1BM93; P23935; P42028
|
GO:0003746
|
translation elongation factor activity
|
1
|
0.030281
|
P43896
|
GO:0051539
|
4 iron, 4 sulfur cluster binding
|
1
|
0.037156
|
P42028
|
GO:0016620
|
oxidoreductase activity, acting on NADP as acceptor
|
1
|
0.043989
|
E1BM93
|
Functional analysis on up-regulated proteins which were specifically activated by MB infection
In the present study, to identify proteins only associated with MB-infection in BAM, protein expression profiles of three treatment (MTB-infection, MB-infection and normal BAM) were compared together. The hierarchical clustering of these up-regulated proteins
showed that all of these significantly up-regulated proteins has similar trends that all these 51 up-regulated proteins were invoked to over-express only in MB-infected BAM (Fig. 5C; Table 5).
Subsequently, Gene Ontology enrichment analysis was carried out on all these significantly up-regulated proteins to elucidate the affected biological processes during the progress of pathogenic bacteria MB infection (Fig. 5A, B; Table 4). For cellular component, a series of corresponding categorizes relevant to energy metabolisms, autophagy and inflammation were significantly identified, including mitochondrial respiratory chain complex II, succinate dehydrogenase complex (ubiquinone), respiratory chain complex II, phagolysosome, macrophage migration inhibitory factor receptor complex, phagolysosome membrane, autolysosome, NLRP3 inflammasome complex, AIM2 inflammasome complex and autophagosome (Fig. 5A; Table 4). It suggested that MB infection could induce various changes in energy metabolisms, autophagy and inflammation of BAM. Additionally, three molecular functions relevant to oxidation-Reduction reactions were overrepresented among these 51 up-regulated proteins, including peroxidase activity, oxidoreductase activity and antioxidant activity (Fig. 5B; Table 4). As imperative progresses, peroxidase activity, oxidoreductase activity and antioxidant activity perform important functions in BAM to defend pathogenic bacteria attacks, such as superoxide dismutase A[20]. Remarkably, in biological progress categorizes, several terms associated with energy metabolisms, autophagy, defense response to bacteria and immune response were dramatically activated in BAM by MB infection, including defense response, hydrogen peroxide-mediated programmed cell death, immune response, mitochondrial electron transport, succinate to ubiquinone, negative regulation of inflammatory response, negative regulation of response to external stimulus, regulation of natural killer cell mediated immunity, respiratory burst after phagocytosis and response to wounding (Fig. 5B; Table 4). All these GO enrichment analysis suggested that activation of energy metabolisms, autophagy, defense response to bacteria and immune response was the main response of BAM under pathogenic bacteria MB attacks.
As the database for protein orthologous classification, Cluster of Orthologous Groups of proteins analysis could contribute the understand of protein functions. We further analyzed these 51 up-regulated proteins with COG protein database to identify their specific function, the results showed that these proteins mainly involved in 17 of 25 KOG categories (Fig. 5E). The highest frequency of occurrence of KOG terms are Signal transduction mechanisms, Intracellular trafficking, secretion, and vesicular transport and Energy production and conversion (Fig. 5E). And the term Defense mechanisms was also involved among these MB-activated proteins (Fig. 5E). Moreover, many identified proteins are involved in Posttranslational modification, protein turnover, chaperones and Carbohydrate transport and metabolism, indicating these functional classifications were also perform some functions in BAM in response to MB infection (Fig. 5E).
Furthermore, according to KEGG pathway database analysis on these 51 proteins, the main biochemical metabolism and signal transduction pathways of BAM in response to MB infection has been described (Fig. 5D). The results indicated that all annotated proteins were significantly mapped onto 18 KEGG pathways, especially Lysosome, Tuberculosis, Phagosome, Apoptosis, mTOR signaling pathway and Autophagy (Fig. 5D). Remarkably, Tuberculosis which caused by MB infection was also significantly identified with Pvalue of 0.032 (Fig. 5D). Additionally, biological progresses relevant to Autophagy and Apoptosis were still the major categorizes, which performed important function in defense response of BAM to pathogenic bacteria MB. And it has been proved that Lysosome and mTOR signaling pathway also perform important functions in defense of macrophage to MTB and MB infection[8–12].
Ultimately, we concluded that MB infection could cause changes in expression of proteins which involved in energy metabolism, autophagy, apoptosis, lysosome and inflammation. And the activation of these terms may be the major progresses of BAM to defend pathogenic MB attacks.
Table 4
Significantly enriched Gene Ontology terms only in MB-infected bovine alveolar macrophage
GO ID
|
Description
|
out (44)
|
pvalue
|
GO:0016209
|
antioxidant activity
|
2
|
0.041502
|
GO:0044754
|
autolysosome
|
1
|
0.042823
|
GO:0005776
|
autophagosome
|
1
|
0.242651
|
GO:0016338
|
calcium-independent cell-cell adhesion via plasma membrane cell-adhesion molecules
|
1
|
0.014941
|
GO:0006952
|
defense response
|
7
|
0.030843
|
GO:0008626
|
granzyme-mediated apoptotic signaling pathway
|
1
|
0.014941
|
GO:0010421
|
hydrogen peroxide-mediated programmed cell death
|
1
|
0.029663
|
GO:0006955
|
immune response
|
7
|
0.043833
|
GO:0036481
|
intrinsic apoptotic signaling pathway in response to hydrogen peroxide
|
1
|
0.029663
|
GO:0006121
|
mitochondrial electron transport, succinate to ubiquinone
|
2
|
0.001285
|
GO:0005749
|
mitochondrial respiratory chain complex II
|
2
|
0.000609
|
GO:1900016
|
negative regulation of cytokine production involved in inflammatory response
|
1
|
0.04417
|
GO:0050728
|
negative regulation of inflammatory response
|
2
|
0.022926
|
GO:0032102
|
negative regulation of response to external stimulus
|
3
|
0.016975
|
GO:0072559
|
NLRP3 inflammasome complex
|
1
|
0.042823
|
GO:0004601
|
peroxidase activity
|
2
|
0.017343
|
GO:0032010
|
phagolysosome
|
1
|
0.014478
|
GO:0061474
|
phagolysosome membrane
|
1
|
0.014478
|
GO:0002717
|
positive regulation of natural killer cell mediated immunity
|
1
|
0.014941
|
GO:0097468
|
programmed cell death in response to reactive oxygen species
|
1
|
0.029663
|
GO:0002715
|
regulation of natural killer cell mediated immunity
|
1
|
0.014941
|
GO:1903034
|
regulation of response to wounding
|
4
|
0.010858
|
GO:0045728
|
respiratory burst after phagocytosis
|
1
|
0.029663
|
GO:0045273
|
respiratory chain complex II
|
2
|
0.000609
|
GO:0042060
|
wound healing
|
5
|
0.000627
|
MB-infection induced proteins were involved complex interaction network in bovine alveolar macrophages
Based on the analyses above, 29 proteins were identified as key proteins which were activated in BAM as being challenged by MB infection (Table 5). Among such 29 proteins, 26 proteins were activated by MB infection, whereas only 3 proteins F1MV85, P46168 and A6QP29 were suppressed by MB attacks in BAM (Table 5). Meanwhile, three proteins relevant to defense and autophagy were further verified by qRT-PCR. The results showed that all these three genes encoding these proteins were significantly induced to up-regulate in macrophage following MB infection, which further supported their important function in defending MB infection (Fig. 6C). In the present study, we used STRING tool (version 10; Szklarczyk et al., 2011), which is directed to a database with known and predicted protein-protein interactions, to identify additional protein information for subsequent functional validation of these key MB infection-induced proteins (Fig. 6A). Remarkably, the results depicted that plenty of protein-protein interactions occurred among these 29 key proteins (Fig. 6A). However, for such condition, no proteins was interconnected with these down-regulated proteins (Fig. 6A). On the contrary, in the further co-expression network, we observed a clearly negative correlation between those down-regulated proteins and up-regulated proteins, and forms a complex link between them (Fig. 6B). And these proteins were mainly involved in various energy metabolisms-, autophagy- and immunity-related pathways, including Tuberculosis, Lysosome, Phagosome, Th17 cell differentiation and Oxidative phosphorylation (Table 6). We considered that there was a complex network of proteins, whose expression were altered in BAM as being challenged by MB infection to make defense responses.
Table 5
Key proteins activated by MB-infection
Protein accession
|
Protein description
|
MB/Bovine Ratio
|
MB/Bovine P value
|
Subcellular localization
|
A0A3S5ZPN6
|
Scavenger receptor class B member 2
|
1.688
|
0.043886
|
endoplasmic reticulum
|
A5PJH7
|
LOC788112 protein
|
1.581
|
0.03298
|
extracellular
|
A6H6Y1
|
BOLA-DQA1 protein
|
1.519
|
0.036096
|
peroxisome
|
E1B726
|
Plasminogen
|
22.512
|
0.020516
|
extracellular
|
F1MGW6
|
Uncharacterized protein
|
2.404
|
0.00133542
|
cytoplasm
|
F1MNI5
|
Prostaglandin G/H synthase 2
|
1.612
|
0.039212
|
extracellular
|
F1MX83
|
Protein S100
|
2.48
|
0.039991
|
cytoplasm
|
F1MYR5
|
Nitric oxide synthase
|
1.615
|
0.015842
|
cytoplasm,nucleus
|
F1MZL6
|
V-type proton ATPase subunit H
|
1.705
|
0.037654
|
cytoplasm
|
F1N610
|
Ig-like domain-containing protein
|
1.607
|
0.031422
|
extracellular
|
G5E5L8
|
Uncharacterized protein
|
1.665
|
0.029085
|
mitochondria
|
P09428
|
Interleukin-1 beta
|
4.234
|
0.005355
|
cytoplasm
|
P26779
|
Prosaposin
|
2.484
|
0.023632
|
extracellular
|
P35720
|
Succinate dehydrogenase cytochrome b560 subunit, mitochondrial
|
1.89
|
0.043107
|
plasma membrane
|
P79345
|
NPC intracellular cholesterol transporter 2
|
1.527
|
0.018958
|
extracellular
|
P81287
|
Annexin A5
|
5.042
|
0.021295
|
cytoplasm
|
Q05204
|
Lysosome-associated membrane glycoprotein 1
|
3.394
|
0.012726
|
plasma membrane
|
Q0VCQ6
|
Programmed cell death 10
|
1.544
|
0.008831
|
cytoplasm
|
Q29423
|
CD44 antigen
|
1.62
|
0.018179
|
plasma membrane
|
Q3SZM3
|
Cytochrome b-245 chaperone 1
|
1.552
|
0.034538
|
mitochondria
|
Q3T100
|
Microsomal glutathione S-transferase 3
|
1.838
|
0.004157
|
plasma membrane
|
Q3ZBK5
|
Tumor necrosis factor alpha-induced protein 8-like protein 2
|
2.805
|
0.013505
|
cytoplasm
|
Q6L708
|
Claudin-1
|
1.564
|
0.011168
|
plasma membrane
|
Q8HXK9
|
Apoptosis-associated speck-like protein containing a CARD
|
1.531
|
0.007273
|
cytoplasm
|
Q95123
|
Succinate dehydrogenase [ubiquinone] cytochrome b small subunit, mitochondrial
|
1.736
|
0.028306
|
mitochondria
|
Q9BGI2
|
Peroxiredoxin-4
|
1.619
|
0.044665
|
extracellular
|
F1MV85
|
Polysaccharide biosynthesis domain containing 1
|
0.664
|
0.025944
|
cytoplasm
|
P46168
|
Beta-defensin 10
|
0.262
|
0.05
|
extracellular
|
A6QP29
|
TBC1 domain family member 2A
|
0.662
|
0.0034376
|
cytoplasm
|
Table 6
Key pathways of proteins activated by MB-infection
#term ID
|
term description
|
observed gene count
|
false discovery rate
|
bta05152
|
Tuberculosis
|
6
|
4.27E-06
|
bta04142
|
Lysosome
|
4
|
0.00022
|
bta04145
|
Phagosome
|
4
|
0.0005
|
bta04659
|
Th17 cell differentiation
|
3
|
0.0024
|
bta00190
|
Oxidative phosphorylation
|
3
|
0.0035
|
bta04672
|
Intestinal immune network for IgA production
|
2
|
0.0081
|
bta04657
|
IL-17 signaling pathway
|
2
|
0.0163
|
bta04064
|
NF-kappa B signaling pathway
|
2
|
0.0181
|
bta04668
|
TNF signaling pathway
|
2
|
0.0205
|