Cardiac apoptosis in severe relapsing fever borreliosis

Abstract

Previous studies revealed that the heart suffers significant injury during experimental Lyme and relapsing fever borreliosis when the immune response is impaired (D. Cadavid, Y. Bai, E. Hodzic, K. Narayan, S. W. Barthold, and A. R. Pachner, Lab. Investig. 84:1439-1450, 2004; D. Cadavid, T. O'Neill, H. Schaefer, and A. R. Pachner, Lab. Investig. 80:1043-1054, 2000; and D. Cadavid, D. D. Thomas, R. Crawley, and A. G. Barbour, J. Exp. Med. 179:631-642, 1994). To investigate cardiac injury in borrelia carditis, we used antibody-deficient mice persistently infected with isogenic serotypes of the relapsing fever agent Borrelia turicatae. We studied infection in hearts 1 to 2 months after inoculation by TaqMan reverse transcription-PCR and immunohistochemistry (IHC) and inflammation by hematoxylin and eosin and trichrome staining, IHC, and in situ hybridization (ISH). We studied apoptosis by terminal transferase-mediated DNA nick end labeling assay and measured expression of apoptotic molecules by RNase protection assay, immunofluorescence, and immunoblot. All antibody-deficient mice, but none of the immunocompetent controls, developed persistent infection of the heart. Antibody-deficient mice infected with serotype 2 had more severe cardiac infection and injury than serotype 1-infected mice. The injury was more severe around the base of the heart and pericardium, corresponding to sites of marked infiltration by activated macrophages and upregulation of interleukin-6 (IL-6). Infected hearts showed evidence of apoptosis of macrophages and cardiomyocytes as well as significant upregulation of caspases, most notably caspase-1. We conclude that persistent infection with relapsing fever borrelias causes significant loss of cardiomyocytes associated with prominent infiltration by activated macrophages, upregulation of IL-6, induction of caspase-1, and apoptosis.

FIG. 1.

Cardiac infection, inflammation, and loss of cardiomyocytes in relapsing fever borreliosis. a) Localization of B. turicatae serotype 2 (arrow) next to cardiomyocytes by immunohistochemistry. 400× magnification. b) A sagital H&E-stained heart section from a scid mouse persistently infected with B. turicatae shows inflammation (arrow) mainly at the base of the heart and the great vessels. 20× magnification. An adjacent 400× magnification field shows the inflammation in more detail. c) Masson's trichrome staining in PBS-inoculated mice shows intact cardiomyocytes in the atrium. 400× magnification. d) Masson's trichrome staining shows loss of cardiomyocytes in B. turicatae serotype 2-infected heart atrium (loss of red stain). 400× magnification. e) Immunohistochemistry for the macrophage activation marker F4/80 in a sagital heart section from a scid mouse persistently infected with B. turicatae shows prominent staining (brown color) at the base and periphery of the heart. 20× magnification. The adjacent picture shows activated macrophages at higher magnification (400× magnification). f) Presence of IL-6 mRNA by nonisotopic in situ hybridization (purple color) in a sagital heart section from a scid mouse persistently infected with B. turicatae serotype 2. 20× magnification.

FIG. 2.

Cardiac apoptosis in relapsing fever and Lyme borreliosis. a) Cardiomyocyte with positive TUNEL staining in the nucleus (brown color) of one Igh6^−/− mouse persistently infected with B. turicatae serotype 2. Magnification, 1,000×. b) Apoptotic body (arrow) and expression of Bax (yellow color) in the heart of a scid mouse infected with B. turicatae. 1,000× magnification. c) TUNEL staining in the nucleus of a cardiomyocyte from an immunosuppressed nonhuman primate persistently infected with B. burgdorferi. 1,000× magnification. d) Anti-caspase-1 antibody shows green fluorescence in mononuclear cells from an Igh6^−/− mouse infected with B. turicatae serotype 2. 400× magnification. e) Negative control section shows lack of staining in an area of severe inflammation and cardiac fiber degradation incubated with 2 μg/ml of rat IgG as a negative control. 400× magnification. f) Myocardium from an uninfected Igh6^−/− mouse stained with anti-caspase-1 antibody shows no signal. 400× magnification. g) Periphery of the left ventricle near the atrium from an Igh6^−/− mouse infected with serotype 1 and FITC stained with anticaspase-1 antibody (200× magnification). h) Consecutive section from the same mouse shows red fluorescence staining (TRITC) after incubation with anti-F4/80 antibody, a marker of activated macrophages. i) Double immunofluorescence staining with anti-caspase-1 (FITC) and anti-F4/80 (TRITC) antibodies shows yellow color, indicating colocalization, in the pericardium and membranes from nearby cardiomyocytes.