Evidence for a unique species-specific hypersensitive epitope in Mycobacterium tuberculosis derived cord factor

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Tuberculosis 93 S1 (2013) S88–S93

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Tuberculosis j o u r n a l h o m e p a g e : h t t p : / / i n t l . e l s ev i e r h e a l t h . c o m / j o u r n a l s / t u b e

Evidence for a unique species-specific hypersensitive epitope in Mycobacterium tuberculosis derived cord factor Ashley M. McMullena,b, Shen-An Hwang a, Kelly O’Sheaa,b, Maureen L. Alirua,c, Jeffrey K. Actor*a,c a

Department of Pathology, Medical School, University of Texas-Houston Medical School, Houston, TX Scholarly Concentration in Molecular and Translational Medicine c MD/PhD Program at the Graduate School of Biomedical Sciences at Houston, Houston, TX b



Tuberculosis TDM Trehalose 6,6’-dimycolate Cord factor Hypersensitivity

Presensitization with Mtb–derived trehalose 6,6’-dimycolate (TDM; cord factor) followed by challenge with the same glycolipid species resulted in elicitation of stronger inflammatory responses than when mice were similarly challenged with M. bovis-derived TDM. Mice presensitized to the homologous Mtb-derived TDM demonstrated cachexic over a 6 day period, whereas similarly presensitized mice challenged with the M. bovis-derived TDM, or with emulsion control, did not experience weight loss. Examination of inflammatory responses demonstrated increased lung histopathology in the Mtb-derived TDM challenged group, evidenced by severe tissue disruption, cellular influx, vascular occlusion and lymphocytic cuffing, and endothelial cell damage. Histological analysis demonstrated that lung pathology in the M. bovis challenged group was strikingly similar to that of the acute model challenge. Examination of proinflammatory mediators also showed findings consistent with histological manifestation, with significantly elevated TNF-D and IL-1E, as well as IFN-J, in the homologous TDM challenged group relative to all other groups. Overall, these findings indicate a difference in hypersensitive immune responses to TDM derived from different mycobacterial strains. Development of specific adaptive immune responses to the Mtb-derived TDM were demonstrated that had limited cross-reactivity to that of M. bovis, thus strongly suggesting the presence of hypersensitive epitopes exclusive to Mtb TDM not present on M. bovis-derived TDM. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis (TB). Trehalose 6,6’-dimycolate (TDM), an abundant glycolipid on the Mtb cell surface, functions as a key mediator of pathogenesis. Recent hypotheses link TDM immune reactivity to development of post primary tuberculosis.1,2 Critical to these hypotheses is the involvement of an adaptive response which contributes to formation of the hypersensitive granuloma. As such, the mouse model of TDM hypersensitivity provides an excellent opportunity to examine the role of adaptive immunity in granulomatous pathology development.3 Multiple laboratories have examined this long chain fatty acid glycolipid for its abilty to induce innate granulomatous responses.4 These investigators have primarily focused on structure function relationships,5-7 on cytokine production, or stress-related factors which influence underlying reactivity.8-10

* Corresponding author at: Department of Pathology and Laboratory Medicine, MSB 2.214, University of Texas-Houston Medical School, 6431 Fannin, Houston, TX 77030, USA. Tel.: 713 500 5344; Fax: 713 500 0730 E-mail address: [email protected] (J.K. Actor). 1472-9792/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.

The induction of an adaptive lymphocytic response to TDM was demonstrated as differentially manifested compared to innate “foreign-body-type” granulomas, with histologic features resembling more advanced stages of tuberculosis pathologies.3 This induced “hypersensitive” granulomatous pathology has been further characterized, with demonstration of a requirement for CD4+ T cells and a contribution by lipid presentation molecules.11-13 The induced hypersensitive granuloma therefore represents a model of a transitional state where adaptive components against glycolipid moities may be further studied. A targeted immune response towards TDM may be critical for development of protective host immunity. Current immunization efforts utilize the bacille Calmette–Guérin (BCG) vaccine, a live attenuated strain of M. bovis. Unfortunately, pathological protection provided by BCG is highly variable; while documented to protect against childhood disease, it does not protect against adult primary infection or latent pulmonary reactivation.14-16 Understanding reactivity to unique glycolipid determinants may assist in understanding deficiencies in the BCG vaccine to protect against virulent Mtb infection. This is the first study to determine if differences between TDMs derived from mycobacterial species M. bovis and Mtb contribute to hypersensitive responsiveness. As such, phenotypic responses were identified which indicate differences exist

A.M. McMullen et al. / Tuberculosis 93 S1 (2013) S88–S93

between cord factor derived from the two species. This report therefore forms the basis for future studies aimed at revealing the unique molecular determinants responsible for elicitation of the Mtb-specific TDM hypersensitive activity. 2. Materials and Methods 2.1. Induction of the TDM Granulomatous Response Mtb derived TDM (cord factor) was purchased from Santa Cruz Biotechnology. M. bovis (Bovine Kidney AN5) derived TDM was purchased from Sigma. Complete and Incomplete Freund’s Adjuvant was purchased from Difco Laboratories. The hypersensitive granulomatous TDM model was performed as described.3,13 BALB/c mice were subcutaneously immunized with Mtb TDM (100Pg) with Complete Freund’s Adjuvant (CFA) as previously described and IV challenged 4 weeks later with Mtb- or M. bovis-derived TDM w/o/w emulsion. For the challenge, mice were injected intravenously via tail vein with 100 PL of emulsion, prepared by dissolving 100 Pg purified TDM in 9:1 hexane/ethanol followed by evaporation of the solvent. TDM was homogenized with Incomplete Freund’s Adjuvant (IFA) (3.2% v/v) and equal volume of 1x phosphate buffered saline (PBS) (Mediatech, Herndon, VA), forming the water/oil emulsion. The outer aqueous layer (93.6% v/v), consisting of 1xPBS with and 0.2% Tween 80 (Mallinckrodt, Hazelwood, MO), was added and homogenized, forming the water/oil/water emulsion. Mice were sacrificed at days 0, 3 and 6 post-TDM challenge. Details for the acute model of TDM granuloma development have been previously described.8,10,17 Briefly, naïve/unsensitized mice were challenged with either TDM species (100 Pg/mouse) and sacrificed after 6 days.


Table 1 Weight change over time in mice senstitized to Mtb-derived post TDM challenge. Animal Weight (grams)


Emulsion Alone

Immunized M. bovis Challenge

Immunized Mtb Challenge

18.52 +/- 1.3 18.81 +/- 1.4 19.35 +/- 1.3 18.62 +/- 1.2

18.70 +/- 1.2 18.91 +/- 1.0 19.05 +/- 0.8 18.29 +/- 1.1

20.00 +/- 0.6 18.92 +/- 1.9 19.62 +/- 0.6 19.07 +/- 0.7

19.36 +/- 1.3 16.29 +/- 1.3 15.17 +/- 1.2* 14.92 +/- 1.2*

Condition Day 0 Day 2 Day 5 Day 6

Mean weight in grams shown for groups, with standard deviation. N=4 for control groups, and 6-8 mice per experimental group. *p
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