Immunomodulatory Effects of Lactoferrin during Burkholderia thailandensis Infection

Melioidosis is a tropical infection caused by the Gram-negative bacterium and Tier 1 select agent Burkholderia pseudomallei. Agnostic whole exome sequencing of DNA from patients with melioidosis has identified single nucleotide variants in the lactoferrin gene that are associated with death. Lactoferrin is a glycoprotein with both antimicrobial and immunomodulatory properties. We investigated whether iron-saturated lactoferrin altered Burkholderia growth or cellular responses to Burkholderia infection. (1) Burkholderia thailandensis, a bacterium closely related to B. pseudomallei and does not require BSL-3 select agent containment, was cultured in liquid M9 minimal media in the presence of varying concentrations of iron-saturated purified human lactoferrin (phLTF) or recombinant human lactoferrin (rhLTF). Growth was quantified at 24 hours. (2) A human monocyte cell line (THP-1) was differentiated and infected with B. thailandensis at multiplicities of infection (MOI) 1 and 10. phLTF or rhLTF 100 μg/mL was added 1 hour after infection and cell supernatants were collected at 6 hours. TNF-α, IFN-γ, and IL-1β concentrations were measured by ELISA. Under these conditions, a kanamycin protection assay was performed 1 hour after infection and at 6 hours, lysed cell supernatants were incubated over 24 hours and colonies counted. (1) B. thailandensis growth in minimal media broth was not altered by the presence of phLTF or rhLTF. (2) Infected THP-1 cells released higher concentrations of IL-1β in the presence of phLTF at MOI 10. Infected THP-1 cells released higher concentrations of TNF-α in the presence of rhLTF at MOI 1 and in the presence of rhLTF or phLTF at MOI 10 (Figure 1, * P < 0.05). IFN-γ concentrations were below the level of detection of the assay. Cells exposed to phLTF expressed more intracellular bacteria compared with controls (Figure 2, * P < 0.05). While lactoferrin does not alter B. thailandensis growth in liquid media, it does increase the release of the pro-inflammatory cytokines in monocytes. Together, these novel results suggest that lactoferrin may have an important immunomodulatory role in melioidosis. Further studies will evaluate the in vitro effect of iron-desaturated lactoferrin and lactoferrin gene variants on microbial activity. T. E. West, NIH: Grant Investigator and Scientific Advisor, Research grant. DTRA: Grant Investigator, Research grant