Toll-like receptor 4 limits transmission of Bordetella bronchiseptica

Abstract

Transmission of pathogens has been notoriously difficult to study under laboratory conditions leaving knowledge gaps regarding how bacterial factors and host immune components affect the spread of infections between hosts. We describe the development of a mouse model of transmission of a natural pathogen, Bordetella bronchiseptica, and its use to assess the impact of host immune functions. Although B. bronchiseptica transmits poorly between wild-type mice and mice lacking other immune components, it transmits efficiently between mice deficient in Toll-Like Receptor 4 (TLR4). TLR4-mutant mice were more susceptible to initial colonization, and poorly controlled pathogen growth and shedding. Heavy neutrophil infiltration distinguished TLR4-deficient responses, and neutrophil depletion did not affect respiratory CFU load, but decreased bacterial shedding. The effect of TLR4 response on transmission may explain the extensive variation in TLR4 agonist potency observed among closely related subspecies of Bordetella. This transmission model will enable mechanistic studies of how pathogens spread from one host to another, the defining feature of infectious disease.

Figure 1. TLR4 is required to prevent transmission of B. bronchiseptica between mice.

The number of CFU of B. bronchiseptica recovered from the nasal cavity of HeJ, HeN, C57BL/6 , μMT, TCR^−/−, or Rag^−/− secondary mice dissected after three weeks of exposure to an index case. Index cases were of the same genotype as secondary mice. Individual secondary mice are represented by dots. Box spans the interquartile range, and the median value is represented by a bar. The whiskers span the 1st and 4th quartile. The limit of detection is marked by a dashed line. The proportion of infected mice in each group is listed above the box and whisker plot. Figure represents the cumulative results of 2–3 independent experiments for each mouse genotype.

Figure 2. Immunodeficiency does not affect resistance to experimental infection with B. bronchiseptica.

Mice were inoculated with 5B. bronchiseptica in 5 µl of PBS and dissected 7 days later. The quantity of B. bronchiseptica recovered from the nasal cavity of individual mice is represented by dots. Box spans the interquartile range with the line denoting the median value. Whiskers span the 1st and 4th quartile. The limit of detection is marked by a dashed line. The proportion of infected mice in each group is listed above the box and whisker plot. Figure represents the cumulative results of 2 independent experiments for each mouse genotype.

Figure 3. TLR4 is required to limit the growth of B. bronchiseptica within the respiratory tract.

3–4 mice per group were examined at 3, 7, 14, and 28 days after inoculation. The bacterial loads of B. bronchiseptica recovered from the (A) nasal cavity or (B) lungs of wild-type (dashed lines) HeN (white squares) or C57BL/6 (black squares), and immunodeficient mice (solid lines): HeJ (black diamonds), μMt (black circles) or Rag^−/− (black triangles). (C) Shedding was detected throughout the infectious course by quantitative culture of bacteria obtained in a ten second swab of the external nares. Symbols represent the mean log₁₀ CFU of B. bronchiseptica (± SEM) Dashed line represents the limit of detection. Figure represents 1 of 2 independent iterations of this experiment.

Figure 4. Intense leukocyte and neutrophil recruitment to the nasal cavity is observed in HeJ but not HeN mice.

Symbols represent the mean ± standard error of flow cytometry-derived counts of white blood cell types: (A) Total leukocytes (CD45⁺), (B) neutrophils (CD11b⁺/Ly6G⁺), (C) macrophages (F4/80⁺/MHCII⁺), (D) NK cells (NK1.1⁺), (E) B cells (B220⁺/MHCII⁺), and (F) T cells (CD3⁺) recovered from the nasal cavity of mice immediately before (day 0) and 1, 3 and 7 days after inoculation of HeJ (black diamonds) or HeN (white squares) mice with 100 CFU of B. bronchiseptica. Experiments were carried out with 4 mice per group in 2 separate iterations.

Figure 5. Depletion of neutrophils reduces shedding from HeJ mice and does not affect within host colonization.

(A) HeN or (C) HeJ mice were given an intraperitoneal injection of 0.5 mg of either isotype control IgG2b (HeN white squares; HeJ black diamonds) or anti-GR1 (black crosses) antibodies prior to infection and every other day thereafter. Shedding was detected by quantitative culture of bacteria obtained in a ten second swab of the external nares. Symbols represent the mean log₁₀ CFU of B. bronchiseptica ± the standard error. The number of CFU of B. bronchiseptica recovered from the nasal cavity, trachea, lung, liver, and spleen of (B) HeN or (D) HeJ mice treated with either isotype control IgG2b (HeN white bars; HeJ black bars) or anti-GR1 antibodies (striped bars) 7 days after inoculation. Bars represent the mean log₁₀ CFU of B. bronchiseptica ± SEM. Dashed line represents the limit of detection. Experiments were carried out with 4 mice per group in 2 separate iterations.

Figure 6. B. bronchiseptica recovered from secondary mouse nasal cavity.

CFU of B. bronchiseptica were recovered from the nasal cavity of HeN secondary mice three weeks after exposure to a HeJ index case (left) or HeJ secondary mice four weeks after exposure to a HeN index case (right). Individual secondary mice are represented by dots. Box and spans the interquartile range, while the whiskers span the 1st and 4th quartile. The proportion of infected mice in each group is listed above the box and whisker plot. Dashed line represents the limit of detection. Figure represents cumulative findings of 2 iterations of the same experiment using 4–5 mice per group.

Figure 7. Naturally transmitted B. bronchiseptica infections begin in the nasal cavity before growing and spreading to the lower respiratory tract.

Index HeJ mice were inoculated with 100(A) nasal cavity, (B) trachea, or (C) lungs of individual recipient HeJ mice after being housed with an index case for 5, 7, 10, 12, and 14 days. The dashed line indicates the limit of detection. Graph represents findings of a single experimental iteration.