<I>Legionella pneumophila</I> mediated activation of dendritic cells involves CD14 and TLR2

Abstract

In this study, we analyzed the activation of bone-marrow derived dendritic cells (BMDCs) from mice lacking the cd14-gene with purified Legionella pneumophila lipopolysaccharide and with viable or formalin-killed L. pneumophila. We found that low concentrations of LPS and doses of L. pneumophila that are relevant to infection are dependent on CD14 to activate BMDCs. Higher concentrations of LPS are able to overcome the lack of CD14 indicating that other receptors areinvolved. We, therefore, included studies using BMDCs from mice lacking functional TLR2 and/or TLR4 molecules. We found that purified L. pneumophila LPS as well as L. pneumophila either viable or formalin-killed are able to activate BMDCs from TLR4-deficient C3H/HeJ mice but fail to activate BMDCs from TLR2-knockout mice. Our data show that not only purified LPS from L. pneumophila but also the microorganism itself stimulate BMDCs via TLR2 and that this stimulation is dependent on CD14 in this mouse model.

Human macrophages utilize a wide range of pathogen recognition receptors to recognize Legionella pneumophila, including Toll-Like Receptor 4 engaging Legionella lipopolysaccharide and the Toll-like Receptor 3 nucleic-acid sensor

“…Epidemiological studies had found that polymorphisms in the human TLR4 gene either predispose or restrict individuals from developing Legionnaires' disease [41,45], and our results suggest that one explanation for this may be variations in the ability of macrophages to recognize infecting legionellae via TLR4. We further demonstrated that human TLR2, TLR5, and their adaptor MyD88 are also required for an optimal cytokine response; this result, unlike the one involving TLR3 and TLR4, does align with prior murine-based studies [26,29,31,33,43,49,51,56]. While examining these TLRs, we also confirmed that the adaptors TRAM and TRAF6 and the receptor CD14 are critical for the cytokine response of human macrophages to L. pneumophila.…”

“…Pertinent to the last scenario, murine and human TLRs do differ significantly in the sequence of their antigen binding domains, and therefore they can differ in their ability to bind like PAMPS [116,118]. Indeed, this might explain why past studies implicated TLR2 (not TLR4) as the murine PRR for L. pneumophila LPS [26,31,33]. In support of there being a possible difference in the delivery or spread of PAMPs, we previously observed that TRIF/TRAM adaptors.…”

“…Third, four serogroups of L. pneumophila, which differ in their O-antigen [103], triggered a similar TLR4-dependent cytokine response, implying that this part of the LPS is not engaging TLR4. Though finding that L. pneumophila LPS is sensed by human TLR4 is not surprising when considering the many other bacteria that have been similarly characterized, it is a major change for the L. pneumophila field, since, as noted above, the long-standing view has been that L. pneumophila LPS is recognized by TLR2 and that TLR4 is not important, as determined from murine models [13,18,25,26,28,31,33,34,117]. While LPS is the canonical PAMP for TLR4, there are other TLR4-activating signals, including glycoinositolphospholipids, lipopeptidophosphoglycans, mannuronic acid, and endogenous danger-associated molecular patterns [24,110,116,118].…”

“…Others, using receptor-knockout mice or macrophages from such mice, reported that TLR3 and TLR4 are not involved in murine-sensing of L. pneumophila [26][27][28][29][30][31][32]. Also, the murine PRR that engages L. pneumophila LPS was described to be TLR2, rather than TLR4, which is most often the PRR linked to LPS [26,31,33,34]. These various findings have regularly appeared in reviews in the field [5,13,14,18,21,25].…”

“…They were then exposed to either depleted zymosan (DZ) or E. coli LPS for 12 h (left panel), or infected for 9, 12, 24, or 48 h with strain 130b (Lpn) inoculated at a MOI 20 (for 9-h infection; left panel) or 0.5 (for 12, 24, and 48-h infections; right panel), and levels of secreted IL-6 (top) and TNFα pneumophila LPS is a critical PAMP that is recognized by human macrophages and specifically the one engaging human TLR4. Although LPS is widely known to be the PAMP recognized by TLR4 following infection by a range of Gram-negative pathogens [17,20,24], prior experimental assessments of L. pneumophila on this particular topic were murine-based and they had concluded that TLR2 is the receptor responding to L. pneumophila LPS [31,33,34,43,49]. Therefore, we used our newly-made panel of KO U937 cells to look directly at how L. pneumophila LPS is recognized by human macrophages.…”