Different cytokine response of primary colonic epithelial cells to commensal bacteria

Table 1 PCR primer sequences.

Gene	Forward 5'-3’	Reverse 5'-3’
Cytokines
IL-1b	GAGATTGAGCTGTCTGCTCA	AAGGAGAACCAAGCAACGAC
IL-6	GATGCTACCAAACTGGATATAATC	GGTCCTTAGCCACTCCTTCTGTG
IL-18	ACTGTACAACCGCAGTAATACGG	AGTGAACATTACAGATTTATCCC
TNFa	TGGGAGTAGACAAGGTACAACCC	CATCTTCTCAAAATTCGAGTGACAA
ENA78 (CXCL5)	CTTCCTCAGTCATAGCCGCAAC	ATCCGTGGGTGGAGAGAATCAG
IP-10 (CXCL10)	TTTCTGCCTCATCCTGCTGG	GGAGCCCTTTTAGACCTTTTTTGG
MIP-1a (CCL3)	GCCCTTGCTGTTCTTCTCTGT	GGCATTCAGTTCCAGGTCAGT
MIP-1b (CCL4)	ACACCATGAAGCTCTGCGT	CGCTGGAGCTGCTCAGTTC
MIP-2a (CXCL2)	CAAAGGCAAGGCTAACTG	TGTTCTACTCTCCTCGGT
MIP-3a (CCL-20)	GCAGAAGCAGCAAGCAACTACG	GAGGTTCACAGCCCTTTTCACC
MCP-1 (CCL2)	CTCACCTGCTGCTACTCATTC	GCTTGAGGTGGTTGTGGAAAA
MCP-3 (CCL7)	TGTGCCTGAACAGAAACCAACC	AAAAATGGGGAAAGGGGGAG
KC (GRO)	GTCCTTTGAACGTCTCTGTC	GCTGGCTTCTGACAACACTA
RANTES (CCL5)	CAAGGAGTATTTCTACACCAGCAGC	ATGCCGATTTTCCCAGGACC
MEC (CCL-28)	GCTGTGTGTGTGGCTTTTCAAAC	TTCTGTCCTTCCTGCTGGGTTG
TLR- signaling
MyD88	ACCCCACTCGCAGTTTGTTGGATG	TGGTGATGCCTCCCAGTTCCTTTG
TIRAP	CAATCTACCTGGAATCGGCTGTC	GCATCTTCTTGGGCTTCTTCAAC
IRAK	TGCCGCTTCTACAAAGTGATGGACG	AATGGGTCTGGGAGCCTGGAAAAG
Caspase-1	ATTACTGCTATGGACAAGGCACGG	CCCTCGGAGAAAGATGTTGAAACTC
TRAF-6	AATGGAAGCACAGCAGTGTAACGGG	AAGGCAAGCAGTTCTGGTTTGGCG
IKKb	TGGAGCCTGGGAAATGAAAGAACG	TCTAAGAGCCGATGCGATGTCACTC
IKKa	AGCCATTCCTCACTTACCAGTCCG	CAGCAGTCTCTTCCACAGAAAACCC
Others
CD45	CTTCAGAGCCTCGTACCAGC	TGTGTCCAGAAGGGCAAATC
b-actin	GCTTCTTTGCAGCTCCTTCGTTG	TTCTCCATGTCGTCCCAGTTGG
Vimentin	CTGTGTCCTCGTCCTCCTACC	GCAGTTCTACCTTCTCGTTGG
b-defensin3	GCATTTGAGGAAAGGAACTCCACAAC	GTCTCCACCTGCAGCTTTTAGCAA
Angiogenin4	CTCTGGCTCAGAATGTAAGGTACGA	GAAATCTTTAAAGGCTCGGTACCC
SAA-1	GTAATTGGGGTCTTTGCC	TTCTGCTCCCTGCTCCTG

Table 2 Semi-quantitative assessment of bacteria-induced CEC gene expression.

Gene	Medium	+B. ovatus	+E. coli	+L. rhamnosus
IL-1β	-1	12	1	3
TNFα	-	0	2	1
IL-6	+	4	2	0
IL-18	0	0	0	0
ENA-78 (CXCL5)	0	1	1	1
IP-10 (CXCL10)	+	1	2	1
MIP-1α (CCL3)	-	0	3	0
MIP-1β (CCL4)	-	0	4	0
MIP-3α (CCL-20)	-	1	3	1
MCP-1 (CCL2)	+	2	4	4
MCP-3 (CCL5)	+	1	2	2
KC (GRO)	+	1	4	2
MIP-2α (CXCL2)	+	0	0	2
RANTES (CCL5)	-	0	2	1
MEC (CCL-28)	-	0	2	1
β-Defensin3	-	0	4	0
SAA-1	-	0	0	0
Angiogenin4	-	0	0	0

¹Absence (–) or presence (+) of EtBr-stained PCR amplicons in CECs cultured in media alone.

²Relative intensity of amplicons expressed by CEC cultured in presence of bacteria compared to cultured in media alone as determined by scanning densitometry. 0 = No change, 1 = <2-fold higher, 2 = 2- to 3-fold higher, 3 = 3- to 4-fold higher, 4 = >4-fold higher.