Cytokine Profile in Ileal and Colonic Tissue of Pigs Is Modulated by Different Fiber Sources with and Without Multi‐Carbohydrase Enzyme Supplementation
Authors: Vila Marta Ferrandis , Zeng Zhikai , Urriola Pedro E. , Shurson Gerald C. , Saqui‐Salces Milena 
Published: November 2023

IntroductionHigh fiber diets with low fermentability reduce the risk of cardiovascular, metabolic and intestinal diseases. In human and animal diets, grains and their by‐products are the main sources of fiber. Dietary fiber is fermented in the distal intestine resulting in the production of short‐chain fatty acids (SCFA) which have anti‐carcinogenic and anti‐inflammatory effects, modulate the expression of genes related to apoptosis, cell proliferation, differentiation, and cancer development and metastasis. Some of the beneficial effects of dietary fiber may be improved by increasing availability of some fiber compounds such as phenols, which can be achieved through supplementing with certain enzymes. Carbohydrases are currently used as dietary supplements to reduce the undesired effects of high fiber consumption in humans, and to increase nutrient digestibility in animal diets. In vitro studies have shown that the addition of exogenous enzymes increases the production of SCFA. However, it is unknown whether the addition of enzymes has an effect on the intestinal immune response. The purpose of this study was to determine the immune profile of the distal intestine of pigs fed high fiber diets with and without supplemental enzymes.MethodsDiets were supplemented with 100 mg/kg of a mixture of carbohydrase enzymes (E) containing 1,500 U/g xylanase, 1,100 U/g β‐glucanase, 110 U/g mannanse, 35 U/g galactosidase. Fifty‐four pigs (25.33 ± 0.41 kg) were randomly assigned to 1 of 6 dietary treatments: 1) standard corn‐soybean meal (CSB, 8% neutral detergent fiber, NDF), 2) CSB + 40% dried distillers grains with solubles (DDGS, 17% NDF), 3) CSB + 30% wheat middlings (WM, 17% NDF), 4) CSB+E, 5) DDGS+E, and 6) WM+E. After consuming their respective diets for 28 days, pigs were euthanized and ileum and colon samples were collected. Total RNA from tissues was isolated and 500 ng used for cDNA synthesis. Gene expression was determined by qPCR and the ΔCt of INFγ, TNFα, IL1β, IL2, IL4, IL6, IL8, IL10, IL11, IL12p40, IL17A, IL23A and IL25 was calculated in reference to HPRT, GAPDH, and 18s, considering primer efficiency. Statistical significance was calculated using ANOVA and corrected for multiple comparisons using Tukey's test. P < 0.05 was considered significant.Results and DiscussionOur results (Table 1) showed that diets without enzyme supplementation have different inflammatory profiles depending on the fiber source and the tissue. The increase of TNFα in pigs fed DDGS triggered a Th1 response in the colon, while feeding WM induced IL4 and IL25 to result in a Th2 response in the ileum. Enzyme supplemented diets had reduced expression of IL17A in the colon, suggesting that cecal fermentation may have an important role in the Th17 response. Pigs fed DDGS+E and WM+E diets showed increased expression of Th1 cytokines IL1β and IFNγ, and reduced the Th2 cytokine IL11 in ileum and colon. These results suggest that feeding high fiber diets supplemented with enzymes to pigs increases the pro‐inflammatory cytokine profile in the ileum and colon independently of fiber source. Overall, the diet containing WM as main fiber source promoted an anti‐inflammatory status through favoring a Th2 response.Support or Funding InformationNational Pork Board
Gene expression in ileum and colon of pigs fed high‐fiber diets with and without carbohydrase enzyme supplementation1,2

Non‐Supplemented
Supplemented
P‐value3

CSB
DDGS
WM
CSB+E
DDGS+E
WM+E
Diet
Enzyme
Interaction

Ileum

IFNγ
0.06
0.06
0.07
0.07
0.07
0.07
0.53
0.67
0.89

TNFα
97
107.7
95.6
86.3
82.8
101.5
0.83
0.29
0.40

IL1β
0.05c
0.06c
0.06c
0.07b,c
0.08a,b
0.09a
0.03
<0.0001
0.07

IL2
1.95
1.99
1.92
1.94
1.87
1.91
0.60
0.06
0.14

IL4
0.03a,b
0.03b
0.04a
0.03a,b
0.03b
0.03b
0.14
0.01
0.01

IL6
0.02
0.02
0.02
0.02
0.02
0.02
0.35
0.11
0.31

IL8
12.3
12.5
11.7
11.7
11.5
11.4
0.40
0.07
0.73

IL10
1.98
1.99
1.96
1.96
1.92
1.95
0.75
0.08
0.48

IL11
455.8a
435.8a
297.9b
422.4a
196.6c
140.6c
<0.0001
<0.0001
<0.001

IL12p40
0.005
0.005
0.005
0.005
0.006
0.006
0.37
0.31
0.37

IL17A
674.9a
692.4a
590.3a,b
548.3a,b
575.6a,b
486.7b
0.06
<0.01
0.96

IL23A
479.7
502.2
513.2
521.4.
530.5
614.9
0.44
0.17
0.74

IL25
0.13a
0.14a,b
0.17b
0.15a,b
0.16b
0.15a,b
0.02
0.29
0.01

Colon

IFNγ
0.07a,b
0.06a
0.06a
0.06a,b
0.07b
0.08b
0.59
<0.01
<0.01

TNFα
119.2b,c
141.5a
134.2a,b
142.8a
114.5b,c
108.1c
0.20
0.02
<0.0001

IL1β
0.04
0.04
0.05
0.04
0.05
0.04
0.59
0.55
0.19

IL2
1.93
2
1.95
1.97
1.99
1.95
0.06
0.57
0.42

IL4
0.01
0.02
0.02
0.02
0.02
0.02
0.14
0.06
0.30

IL6
0.03
0.03
0.03
0.03
0.03
0.03
0.59
0.33
0.47

1L8
12.2
13.6
12.9
13.3
13.4
13.2
0.11
0.21
0.21

IL10
1.95
2.01
1.96
2.01
2.05
2.01
0.08
0.01
0.98

1L11
480.2a,b
537.5a
532.6a
396.9b,c
342.6c
321.8c
0.80
<0.0001
0.09

IL12p40
0.004
0.004
0.004
0.004
0.005
0.005
0.07
0.17
0.23

IL17A
1295a
1500a
1318a
953b
940b
741b
0.07
<0.0001
0.29

IL23A
785.6
963.1
822.8
1006.1
960.5
976.9
0.33
<0.01
0.08

IL25
0.11
0.11
0.11
0.11
0.12
0.13
0.17
0.01
0.08

Relative expression mean values (n = 9). Different superscripts within each row differ (P < 0.05).
Values are expressed as a relative ratio of the amount of target gene copies to the amount of HPRT, Gapdh, and 18s (housekeeping genes) copies.
The P‐values represent the main effect of the diet, main effect of the enzyme supplementation, and the interaction between the diet and enzyme supplementation.