Lactoferrin is a dynamic protein in human melioidosis and is a TLR4-dependent driver of TNF- release in Burkholderia thailandensis infection in vitro

Author summary Melioidosis is a severe tropical infection caused by the bacteriumBurkholderia pseudomallei. Despite antibiotics, mortality in some regions remains very high, necessitating the need for alternative treatment strategies, including targeting the immune system. Lactoferrin is an iron-binding protein with a variety of different functions. In this study, we wanted to test whether lactoferrin alters how the immune system responds during melioidosis. To achieve this, we first tested the blood of melioidosis patients and found that patients who later died had higher lactoferrin levels compared to those who survived. We also stimulated blood obtained from healthy individuals withB.pseudomalleiand found that lactoferrin levels increase. We next analyzed whether lactoferrin impaired how the bacteria grows and found that the growth ofBurkholderia thailandensis, a closely related bacterium, was not affected by the addition of lactoferrin to the media. When human immune cells, called monocytes, were infected withB.thailandensis, we found that levels of a specific inflammatory protein, TNF-alpha, increased after adding lactoferrin and that this effect was related to a specific immune recognition pathway called Toll-like receptor 4. These findings provide new data about the role of lactoferrin in modulating the immune response in melioidosis. Melioidosis is an often-severe tropical infection caused byBurkholderia pseudomallei (Bp) with high associated morbidity and mortality.Burkholderia thailandensis(Bt) is a closely related surrogate that does not require BSL-3 conditions. Lactoferrin is an iron-binding glycoprotein that can modulate the innate inflammatory response. Here we investigated the impact of lactoferrin on the host immune response in melioidosis. Lactoferrin concentrations were measured in plasma from patients with melioidosis and following ex vivo stimulation of blood from healthy individuals.Btgrowth was quantified in liquid media in the presence of purified and recombinant human lactoferrin. Differentiated THP-1 cells and human blood monocytes were infected withBtin the presence of purified and recombinant human lactoferrin, and bacterial intracellular replication and cytokine responses (tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta and interferon-gamma) were measured. In a cohort of 49 melioidosis patients, non-survivors to 28 days had significantly higher plasma lactoferrin concentrations compared to survivors (median (interquartile range (IQR)): 326 ng/ml (230-748) vs 144 ng/ml (99-277), p<0.001). In blood stimulated with heat-killedBp, plasma lactoferrin concentration significantly increased compared to unstimulated blood (median (IQR): 424 ng/ml (349-479) vs 130 ng/ml (91-214), respectively; p<0.001). Neither purified nor recombinant human lactoferrin impaired growth ofBtin media. Lactoferrin significantly increased TNF-alpha production by differentiated THP-1 cells and blood monocytes afterBtinfection. This phenotype was largely abrogated when Toll-like receptor 4 (TLR4) was blocked with a monoclonal antibody. Lactoferrin is produced by blood cells after exposure toBpand lactoferrin concentrations are higher in 28-day survivors in melioidosis. Lactoferrin induces proinflammatory cytokine production afterBtinfection that may be TLR4 dependent.