Role of Toll-like receptor 1, 2 and 4 on thromboxane B2 production by different microbial products

Background:

Thromboxane A₂ (TXA₂; the stable degradation product is thromboxane B₂) was identified as an important vasoconstrictor upon Kupffer cell (KC) activation via Toll-like receptors (TLRs).

Objectives:

We aimed at investigating the relevant TLRs on KCs activated by different microbial products.

Methods:

THP-1 cell line and primary KC isolated from human tissues (HKC, tissues from HTCR foundation) or mice (MKC) were investigated. Thromboxane B₂ (TXB₂) in supernatants was measured after 24h stimulation by TLR 1 – 9 agonists or bacterial isolates [Klebsiella pneumoniae (KLPN), Escherichia coli (E. coli), Enterobacter cloacae (ENCL), Enterococcus faecium (ENCF), Streptococcus pneumoniae (STPN), Staphylococcus aureus (STAU) and Candida albicans (CAAL), each 8 µg/ml]. The special TLR antagonists (CU-CPT22, 10µM; Mab-mTLR2, 1 µg/ml and TAK-242, 1µM) were additionally investigated.

Results:

All cell types were characterized by immunostaining. Stimulation with microbial products increased TXB₂ efflux in THP-1, HKCs and MKCs (for HKCs see according figure).

TLR1, 2 and 4 agonists also increased TXB₂ efflux on different kinds of KCs: TLR1 149 ± 26 vs. 267 ± 130*, 6380 ± 5357 vs. 127004 ± 150292* and 21 ± 13 vs. 88 ± 58*; TLR2 122 ± 36 vs. 1399 ± 681*, 5724 ± 1649 vs. 281468 ± 358872* and 74 ± 40 vs. 336 ± 199*; TLR4 136 ± 40 vs. 117 ± 28, 8782 ± 6931 vs. 106933 ± 110249* and 48 ± 32 vs. 231 ± 120* pg/ml each in THP-1 cells, HKCs and MKCs. The other TLR agonists had no effect on TXB₂ production.

Additional 1h pretreatment with TLR1, 2 or 4 antagonist in HKCs reduced the TXB₂ increase by E. coli, ENCF and STPN.

Abb. 1: Fig-HKC&bacteria

Conclusions:

This is the first study describing the increase of the vasoconstrictor TXB₂ following activation of human KCs with different, clinically relevant human microbial isolates. These results might improve individual medical treatment e.g. using specific inhibition for TLR 2 on patients with liver diseases and portal hypertension triggered by microbes.