Sensing of Escherichia coli and LPS by mammary epithelial cells is modulated by O-antigen chain and CD14

Abstract

Escherichia coli is one of the major pathogens causing mastitis in dairy cattle. Yet, the factors which mediate the ability for E. coli to develop in the bovine mammary gland remain poorly elucidated. In a mouse model, infections induced by the reference mastitis E. coli P4 showed a strong colonisation of the mammary gland, while this strain had a low stimulating power on cells of the PS bovine mammary epithelial cell line. In order to understand if such a reduced response contributes to the severity of infection, a library of random mutants of P4 strain was screened to identify mutants inducing stronger response of PS cells. Among hyper-stimulating mutants, six were altered in genes involved in biosynthesis of lipopolysaccharide (LPS) and had lost their O-polysaccharide region, suggesting that the presence of O-antigen impairs the response of PS cells to LPS. Using purified smooth (S) and rough (R) fractions of LPS, we showed that the R-LPS fraction induced a stronger response from PS cells than the smooth LPS fraction. Biological activity of the S-LPS fraction could be restored by the addition of recombinant bovine CD14 (rbCD14), indicating a crucial role of CD14 in the recognition of S-LPS by Mammary Epithelial Cells (MEC). When S-LPS and R-LPS were injected in udder quarters of healthy lactating cows, an inflammation developed in all infused quarters, but the S-LPS induced a more intense pro-inflammatory response, possibly in relation to sizeable concentrations of CD14 in milk. Altogether, our results demonstrate that the O-antigen modulates the pro-inflammatory response of MEC to LPS, that S-LPS and R-LPS trigger different responses of MEC and that these responses depend on the presence of CD14.

Leptospiral LPS escapes mouse TLR4 internalization and TRIF-associated antimicrobial responses through O antigen and associated lipoproteins

“…This would be consistent with previous studies showing that smooth LPS (S-LPS) depends on CD14 to activate cells [42,45]. On the other hand, it is established that rough LPS (R-LPS) depends on CD14 mainly for TLR4 endocytosis [24,25], although some studies reported both in vitro and in vivo that R-LPS could benefit from the presence of CD14 [24,46]. Accordingly, several other studies have described R-LPS as a more potent agonist of the innate immune system than S-LPS [42,46–49].…”

“…On the other hand, it is established that rough LPS (R-LPS) depends on CD14 mainly for TLR4 endocytosis [24,25], although some studies reported both in vitro and in vivo that R-LPS could benefit from the presence of CD14 [24,46]. Accordingly, several other studies have described R-LPS as a more potent agonist of the innate immune system than S-LPS [42,46–49]. Overall, such findings are consistent with our results showing that the shorter LPS naturally signals more via TRIF than the full-length LPS, and that sCD14 contributes to this enhanced signaling.…”

Leptospiral LPS escapes mouse TLR4 internalization and TRIF‑associated antimicrobial responses through O antigen and associated lipoproteins

“…This would be consistent with previous studies showing that smooth LPS (S-LPS) depends on CD14 to activate cells [ 40 , 43 ]. On the other hand, it is established that rough LPS (R-LPS) depends on CD14 mainly for TLR4 endocytosis [ 21 , 22 ], although some studies reported both in vitro and in vivo that R-LPS could benefit from the presence of CD14 [ 21 , 44 ]. Accordingly, several other studies have described R-LPS as a more potent agonist of the innate immune system than S-LPS [ 40 , 44 – 47 ].…”

Type 3 immunity: a perspective for the defense of the mammary gland against infections

“…Of note, under these conditions, MECs markedly overexpressed (mRNA) CCL20, a chemokine that attracts cells expressing the receptor CCR6, which include most of type 3 immunity cells [25]. This chemokine was found at the protein level in milk from bovine MGs exposed to E. coli LPS [26].…”