Haemophilus ducreyi partially activates human myeloid dendritic cells

Abstract

Dendritic cells (DC) orchestrate innate and adaptive immune responses to bacteria. How Haemophilus ducreyi, which causes genital ulcers and regional lymphadenitis, interacts with DC is unknown. H. ducreyi evades uptake by polymorphonuclear leukocyte and macrophage-like cell lines by secreting LspA1 and LspA2. Many H. ducreyi strains express cytolethal distending toxin (CDT), and recombinant CDT causes apoptosis of DC in vitro. Here, we examined interactions between DC and H. ducreyi 35000HP, which produces LspA1, LspA2, and CDT. In human volunteers infected with 35000HP, the ratio of myeloid DC to plasmacytoid DC was 2.8:1 in lesions, compared to a ratio of 1:1 in peripheral blood. Using myeloid DC derived from monocytes as surrogates for lesional DC, we found that DC infected with 35000HP remained as viable as uninfected DC for up to 48 h. Gentamicin protection and confocal microscopy assays demonstrated that DC ingested and killed 35000HP, but killing was incomplete at 48 h. The expression of LspA1 and LspA2 did not inhibit the uptake of H. ducreyi, despite inactivating Src kinases. Infection of DC with live 35000HP caused less cell surface marker activation than infection with heat-killed 35000HP and lipopolysaccharide (LPS) and inhibited maturation by LPS. However, infection of DC with live bacteria caused the secretion of significantly higher levels of interleukin-6 and tumor necrosis factor alpha than infection with heat-killed bacteria and LPS. The survival of H. ducreyi in DC may provide a mechanism by which the organism traffics to lymph nodes. Partial activation of DC may abrogate the establishment of a full Th1 response and an environment that promotes phagocytosis.

FIG. 1.

Fluorescence-activated cell sorter analysis of lineage-negative, HLA-DR⁺ cells from H. ducreyi-infected skin (right) and peripheral blood (left) obtained at the time of biopsy from one subject. The numbers in the dot plots represent the percentages of cells in the quadrants.

FIG. 2.

Percentage of DC death after exposure of DC to H. ducreyi (white bars) compared to that for uninfected DC (solid bars) at various time points. Data are expressed as the percentages of annexin V (Ax V)- and/or 7-amino-actinomycin D (7-AAD)-stained cells in the total cell population and represent the means ± SDs for assays performed with DC from four donors.

FIG. 3.

Numbers of H. ducreyi CFU that were internalized and viable in 10⁴ to 10⁵ DC after 90 min of culture, gentamicin treatment, and subsequent culture in medium without antibiotics. The values represent the means ± SDs for assays performed with DC from six donors.

FIG. 4.

Confocal microscopy of DC infected with GFP-expressing H. ducreyi. (A) Optical sectioning of DC that had been incubated with H. ducreyi for 90 min and 24 h. (B) Percentages of DC that had associated (top) or internalized (bottom) bacteria under opsonized (white bars) and nonopsonized (black bars) conditions. The data represent the means ± SDs for assays performed with DC from three donors.

FIG. 5.

Effect of growth phase and expression of LspA1 and LspA2 on H. ducreyi uptake by and Src kinase activation in DC. (A) Percentages of DC containing GFP-expressing bacteria by flow cytometry after incubation with mid-log- and stationary-phase 35000HP/pRB157K or 35000HPΩ12/pRB157K for 90 min. Values are the means ± SDs of the percentages of GFP-positive DC obtained from seven or six donors and cultured with mid-log-phase (MOI of 10) or stationary-phase (MOI of 50 to 100) bacteria, respectively. (B) Western blot of DC incubated with mid-log- or stationary-phase 35000HP or 35000HPΩ12 for 4 h under the conditions described for panel A. Active forms of Src kinases were determined by the α-pY418 antibody. Actin levels were measured as loading controls. Similar results were observed for DC obtained from five donors.

FIG. 6.

Cytokine production by DC exposed to live (LV) or heat-killed (HK) H. ducreyi and LPS singly or in combination after 48 h. Culture supernatants were obtained from DC whose activation levels are reported in Table 1. The levels of IL-6 (A), TNF-α (B), IL-12p70 (C), and IL-10 (D) were assessed by enzyme-linked immunosorbent assay. Data are the means ± SDs of results obtained from six volunteers.