Macrophage p38 mitogen-activated protein kinase activity regulates invariant natural killer T-cell responses during Borrelia burgdorferi infection

Abstract

The interaction of macrophages with infectious agents leads to the activation of several signaling cascades, including mitogen-activated protein (MAP) kinases, such as p38. We now demonstrate that p38 MAP kinase-mediated responses are critical components to the immune response to Borrelia burgdorferi. The pharmacological and genetic inhibition of p38 MAP kinase activity during infection with the spirochete results in increased carditis. In transgenic mice that express a dominant negative form of p38 MAP kinase specifically in macrophages, production of the invariant natural killer T (iNKT) cell-attracting chemokine MCP-1 and of the antigen-presenting molecule CD1d are significantly reduced. The expression of the transgene therefore results in the deficient infiltration of iNKT cells, their decreased activation, and a diminished production of interferon γ (IFN-γ), leading to increased bacterial burdens and inflammation. These results show that p38 MAP kinase provides critical checkpoints for the protective immune response to the spirochete during infection of the heart.

Figure 1.

Inhibition of p38 mitogen-activated protein (MAP) kinase during infection with Borrelia burgdorferi results in increased Lyme carditis. A, Arthritis and carditis in C3H mice infected with B. burgdorferi and treated with the p38 MAP kinase inhibitor SB203580 every other day during infection or in control injected mice (Control). The mice were infected with 10⁵ spirochetes and treated with 1 mg/kg of SB203580. The mice were analyzed after 2 weeks of infection. Ave, average; SE, standard error. B, Representative hematoxylin and eosin histological sections of the hearts of the infected mice. Carditis was scored on the basis of inflammatory infiltrate, including the infiltration of connective tissue with macrophages at the base of the heart, surrounding the aortic valve and the atria.

Figure 2.

Generation of cd11b-dnp38 mitogen-activated protein (MAP) kinase transgenic (Tg) mice. A, Schematic representation of the dnp38 MAP kinase gene used to generate the Tg mice (top), showing the promoter (cd11b), the dnp38 gene, and the human growth hormone polyA/intron signal sequence. The arrows represent the localization of the primers used to detect messenger RNA expression in different tissues (bottom) plus bone marrow–derived macrophages (BMMs). Primers for actin were used as control. B, Percentage of F4/80+ cells in the spleens of Tg and negative littermate control (NLC) mice. Mice aged 6–8 weeks were analyzed for macrophage concentration by flow cytometry upon staining with an anti-F4/80 monoclonal antibody. The analysis was performed on gated live cells according to their forward scatter versus side scatter profile. C, Surface expression levels of CD11b (top) and TLR2 (bottom) in BMMs from Tg mice (grey histograms) and NCL mice (black histograms), determined by flow cytometry. D, Tg and NLC BMMs were stimulated with Borrelia burgdorferi (multiplicity of infection = 25) for 16 hours. The stimulation supernatants were then assessed for interleukin 6 (IL-6) by enzyme-linked immunosorbant assay. *P < .01, by the Student t test.

Figure 3.

Infection of cd11b-dnp38 transgenic (Tg) mice with Borrelia burgdorferi results in increased carditis. A, Arthritis and carditis in 2-week infected cd11b-dnp38 Tg and negative littermate control (NLC) mice. Groups of mice were infected with B. burgdorferi and analyzed for joint and cardiac inflammation. The results represent the average (Ave) ± standard error (SE) of X10 mice in 2 independent experiments. B. burgdorferi levels in the ear (B) and heart (C) of the Tg and NLC infected mice. The results represent the average ± SE of X10 mice in 2 independent experiments. TLR2, Toll-like receptor 2. D, B. burgdorferi–specific immunoglobulin G levels in the sera of Tg and NLC infected mice. Serial dilutions of murine sera were probed in a microtiter plate coated with 0.5 μg/mL of a B. burgdorferi lysate. OD, optical density; UI, uninfected NLC control. The results correspond to 5 mice per group and are representative of 2 experiments. E, Phagocytosis of GFP-expressing B. burgdorferi by BMMs of Tg mice (grey histogram) and NLC mice (black histogram). The cells were incubated at a multiplicity of infection of 10 for 4 hours, followed by washing and flow cytometry analysis. The grey-filled histogram represents the 4°C control.

Figure 4.

Decreased invariant natural killer T (iNKT) cell infiltration and interferon γ (IFN-γ) production in the hearts of infected transgenic (Tg) mice. Quantitative real-time polymerase chain reaction (PCR) of Tg (black bars) and negative littermate control (NLC; white bars) mouse heart messenger RNA (mRNA) for the quantification of vα14i-jα18 NKT cells (A) and ifnγ relative to actin expression (B). C, Relative expression of mcp-1 in the hearts of Tg and NLC infected mice. mRNA was analyzed by quantitative real-time PCR with primers specific for mcp-1. The expression levels were determined relative to those of actin. The results in A–C represent the average ± standard error (SE) of 10 mice per group from 2 independent experiments. *P < .05, by the Student t test. D, Expression of MCP-1 by BMMs from Tg and NLC mice stimulated with B. burgdorferi (Bb; multiplicity of infection = 50) or left unstimulated (Unst.). The results represent the average ± SE of 1 experiment performed in triplicate and are representative of 2 experiments. *P < .05, by the Student t test.

Figure 5.

Decreased expression of CD1d and invariant natural killer T (iNKT) cell activation in cd11b-dnp38 transgenic (Tg) mice. A, Relative expression of cd1d in the hearts of infected Tg and negative littermate control (NLC) mice. Messenger RNA was analyzed by quantitative real-time polymerase chain reaction, and the expression levels of cd1d were calculated relative to actin expression. The results represent the average ± standard error (SE) of 10 mice per group from 2 independent experiments. *P < .05, by the Student t test. B, Surface expression levels of CD1d in BMMs from Tg and NLC mice. BMMs were generated from Tg (grey histogram) and NLC (black histogram) mice and stained with a specific anti-CD1d monoclonal antibody, labeled with PE. The cells were analyzed by flow cytometry. The grey-filled histogram represents the isotype-labeled control. The result is representative of 3 experiments. C, Decreased production of interferon γ (IFN-γ) ex vivo by splenocytes from the cd11b-dnp38 Tg mice (black bar) as compared to NLC mice (white bar) treated with the prototypic iNKT antigen α-galactosylceramide (αGalCer). Groups of 5 mice were injected with 100 ng/g of αGalCer, and 12 hours later whole splenocytes were incubated ex vivo for 72 hours. IFN-γ levels were then determined by enzyme-linked immunosorbant assay. *P < .05, by the Student t test.