Expression of human TLR4/myeloid differentiation factor 2 directs an early innate immune response associated with modest increases in bacterial burden during Coxiella burnetii infection

Abstract

Human TLR4 (hTLR4) and mouse TLR4 molecules respond differently to hypo-acylated LPS. The LPS of Coxiella burnetii is hypo-acylated and heavily glycosylated and causes a minimal response by human cells. Thus, we hypothesized that mice expressing hTLR4 molecules would be more susceptible to C. burnetii infection. Our results show that transgenic mice expressing hTLR4 and the human myeloid differentiation factor 2 (MD-2) adaptor protein (hTLR4/MD-2) respond similarly to wild type mice with respect to overall disease course. However, differences in bacterial burdens in tissues were noted, and lung pathology was increased in hTLR4/MD2 compared to wild type mice. Surprisingly, bone marrow chimera experiments indicated that hTLR4/MD-2 expression on non-hematopoietic cells, rather than the target cells for C. burnetii infection, accounted for increased bacterial burden. Early during infection, cytokines involved in myeloid cell recruitment were detected in the plasma of hTLR4/MD2 mice but not wild type mice. This restricted cytokine response was accompanied by neutrophil recruitment to the lung in hTLR4/MD2 mice. These data suggest that hTLR4/MD-2 alters early responses during C. burnetii infection. These early responses are precursors to later increased bacterial burdens and exacerbated pathology in the lung. Our data suggest an unexpected role for hTLR4/MD-2 in non-hematopoietic cells during C. burnetii infection.

Keywords: Chimera; cytokines; neutrophil infiltration; transgenic.

Figure 1.

Mice expressing human TLR4 (hTLR4)/myeloid differentiation factor 2 (MD-2) have larger spleens and higher spleen C. burnetii burdens early and with lower infectious doses and higher lung bacterial burdens later after higher dose infection. Wild type and hTLR4/MD-2 transgenic mice were infected intratracheally (i.t.) with 10, 100, or 1000 genome equivalents (GE) of C. burnetii Nine Mile phase I (NMI) and sacrificed 6, 9, or 30 d post infection. (a) Spleens were weighed as a marker of disease severity. Bacterial burdens in spleens (b) and lungs (c) were assessed by qPCR. Graphs contain data from experiments conducted at different times. All graphs represent the average of at least four mice per group, with standard error shown. These data are representative of between two and four repeat experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 as measured in pairwise comparisons of two groups using Student’s t-test.

Figure 2.

Chimeric mice demonstrate that non-hematopoietic cells direct enhanced susceptibility in hTLR4/MD-2 mice. The mice expressing hTLR4/MD-2 on stromal/epithelial cells had greater bacterial burdens in the spleens compared to wild type mice, regardless of the type of hematopoietic cells they received. These data are representative of two repeat experiments. **P < 0.01 and ***P < 0.001 as measured by one-way ANOVA with Bonferroni’s multiple comparison test, Error bars represent standard error.

Figure 3.

Mice expressing hTLR4/MD-2 had increased cellular infiltration and pathology in the lung compared to wild type mice. Wild type and hTLR4/MD-2 mice were infected i.t. with 1000 GE of C. burnetii NMI for 9, 30, and 60 d. Lung and spleen tissue sections were stained with hematoxylin and eosin. (a) At all three intervals, hTLR4/MD-2 lung sections show increased infiltration throughout the parenchyma, increased bronchial and vascular inflammation (arrows), and moderate peribronchial and perivascular inflammation (arrowheads) compared to wild type mice. (b) The sections were scored for five parameters on a 0–3 scale, and the average score per group at each interval was graphed. *P < 0.05, **P < 0.01, and ***P < 0.001, as measured in pairwise comparisons of two groups using Student’s t-test, Error bars represent standard error.

Figure 4.

Differences in plasma cytokine concentrations between the two strains were detected early following C. burnetii infection. Significantly more (a and b) CXCL1 (KC) and(c and d) G-CSF was detected in plasma from mice expressing hTLR4/MD-2 compared to wild type mice early after infection (16 or 24 h) with 1000 or 10,000 GE C. burnetii, respectively. (e) By 6 d post infection, in contrast, the detection of G-CSF was lower, and levels were not significantly different between the two strains. All graphs represent the average of at least four mice per group with standard deviation shown. **P < 0.01, ***P < 0.001, and ****P < 0.0001 as measured in pairwise comparisons of two groups using Student’s t-test.

Figure 5.

Early neutrophil influx to the lungs of hTLR4/MD-2 mice. (a) Representative bronchoalveolar lavage (BAL) cytospin images show that at 16 h post infection with 10⁵ GE C. burnetii, mice expressing hTLR4/MD-2 had a notable influx of neutrophils that was not observed in wild type mice and was largely resolved by 40 h post infection. (b) The number of neutrophils was greater but not significantly different at 16 h post infection. This trend was not apparent at later intervals (at least three mice per group). (c) The number of neutrophils at 16 h post infection was not associated with a substantial decrease in macrophages in the BAL. There were no significant differences in BAL macrophages at any interval. Error bars represent standard error.