"Use of Mycobacterium leprae-infected Gene Knockout Mice as Models for the Human Immunopathological Spectrum of Leprosy"
An Interpretive Summary
Linda B. Adams, Ph.D.
DHHS/HRSA/BPHC
Division of National Hansen's Disease Programs
Baton Rouge, La.

  

 

The immunopathological spectrum of leprosy.

Most persons exposed to Mycobacterium leprae will not develop leprosy.  In susceptible individuals an indeterminate lesion may form.  Indeterminate leprosy often spontaneously heals, but it may progress into the leprosy spectrum.  The Ridley-Jopling classification combines clinical, histopathological, and immunological criteria to identify five forms of leprosy: tuberculoid (TT), borderline tuberculoid (BT), mid-borderline (BB), borderline lepromatous (BL), and lepromatous (LL) leprosy.  Another classification scheme was developed by the World Health Organization and was designed for distinct multidrug therapy regimens.  It is made up of two broad categories: paucibacillary (PB) disease, which includes TT and BT, and multibacillary (MB) disease, which includes BB, BL and LL.  Toward the PB end of the spectrum, leprosy lesions have features of a well developed cell mediated immunity (CMI) and contain few acid fast bacilli (AFB), characteristics of a TH1-type immune response.  Toward the MB end of the spectrum, the immune response displays a TH2-type profile with a poorly developed CMI and numerous AFB.  The borderline area of the spectrum is highly unstable and represents poorly understood immunoregulatory responses; BT and BB (and TT) patients are prone to disfiguring reversal reactions (RR) while BL (and LL) patients are subject to painful ENL (erythema nodosum leprosum) reactions.

 

 

Why try to model the human leprosy spectrum?

Because leprosy represents a fascinating model of human immunoregulatory disease, the development of murine models more representative of human leprosy is an important research goal.  Understanding the mechanisms underlying the specific M. leprae anergy in CMI in LL is one goal, but especially worthwhile would be a model of the broad borderline area of the spectrum in which immunological instability often results in RR and ENL reactions.  Understanding the basic mechanisms could lead to means of preventing or predicting these reactions, or of identifying the key components of CMI that need to be stimulated with an effective vaccine.


The advent of gene transfer technology has introduced a new approach in immunology research.  Numerous targeted gene knockout (KO) mouse strains are now commercially available, and include those unable to produce specific cytokines, cytokine receptors, and cluster of differentiation (CD) surface markers.  Studies using such mice have provided a means to evaluate immunological parameters as well as the compensatory mechanisms operational in their absence.  These properties have proven useful in the study of a variety of infectious diseases, and should be especially enlightening in improving our understanding of the pathogenesis of leprosy where a broad clinical spectrum is dependent on the immunological, and perhaps compensatory, responses of the host to the antigens of M. leprae. Our laboratory is exploiting certain KO strains to explore CMI to M. leprae both in vivo and in vitro and to study the microenvironment of the granuloma in leprosy pathogenesis.

 

 

Choice of KO Strains.

The KO strains that we are studying each carry a gene deletion that is considered important in the host CMI response to intracellular pathogens at the level of the macrophage or T cell effector function. We are also interested in genes which are important in immunoregulation in the localized microenvironment of the granuloma.  Several KO mouse strains are under investigation:

 

IFNγ KO

 T and NK cells are unable to make interferon-gamma, a key cytokine responsible for macrophage activation.

ROI KO

 model for X-linked chronic granulomatous disease [X-CGD]; phagocytic cells are unable to generate a respiratory burst.

RNI KO

 activated macrophages cannot produce nitric oxide synthase which generates toxic nitrogen radicals.

nu/nu

 congenitally athymic, hairless mice which do not generate functionally mature T cells.

CD4 KO

 these mice do not have functional CD4+ T cells, a regulatory T cell and the same cell subset which is depleted in AIDS.

CD8 KO

 this strain does not have functional CD8+ T cells, a cytotoxic cell which can lyse infected macrophages.

IL-12 KO

 this strain of mice do not generate interleukin-12, an important regulatory cytokine of the immune system which induces the production of IFN-γ by T cells and NK cells and stimulates the development of a TH1 type response.

IL-10 KO

 these mice are deficient in interleukin-10, a cytokine which is generated by T cells and macrophages and is an inhibitor of IFN-γ production. 

TNFR KO

the cells in these mice do not have a tumor necrosis factor (TNF) receptor and are therefore unresponsive to TNF.  TNF is a cytokine which has been shown to be important in granuloma formation.
 

 

Study Parameters

In these studies, we are exploring the outcome of M. leprae foot pad infection in carefully chosen KO mouse strains which represent impediments to both acquired and innate immunity.  Bacterial growth is monitored and detailed analyses on the experimental foot pad granulomas, such as cellular profile and cytokine production, are being conducted. The findings will help to determine if models of infection which can be classified along the leprosy spectrum can be developed.

 

 

Initial Findings

RNI KO - This strain of mice exhibits large, organized granulomas, composed of numerous epithelioid cells, a few macrophages, and dense collections of lymphocytes.  These features, along with a strong TH1 cytokine response and the limitation of M. leprae growth, resemble borderline tuberculoid leprosy.

IFNγ KO - These mice exhibit augmented but not uncontrolled growth of M. leprae, enhanced but not well-organized cellular infiltration into the foot pad with only scattered lymphocytes present, and the presence of TH2 type cytokines.  These are attributes present in mid-borderline leprosy.

nu/nu - Early studies in the 1970's on this strain defined the role of T cell CMI responses in leprosy.  Local foot pad multiplication of M. leprae appears to be unchecked, reaching 1 x 1010 bacilli or more.  Histologically, the granuloma consists of foamy macrophages which contain enormous globi of bacteria.  These features resemble lepromatous leprosy.

 

 

Further Modification of KO status.

Where appropriate, we are further modifying CMI responses in these KO mice by conditionally knocking-out additional CMI gene functions before or after infection with M. leprae or selectively restoring certain disrupted gene functions after infection.  These modifications could induce a change in the disease presented (i.e. Adowngrading@ or Aupgrading@ in the position on the spectrum, or a reactional state) and may provide clues for the mechanisms underlying destabilization of the status quo.

 

 

The ultimate goal of these proposed studies is to advance our basic knowledge of the role of the CMI response in host resistance to M. leprae.  These findings may also provide fundamental insights toward the prevention of nerve damage in leprosy, furnish a focus for vaccine improvement and the development of efficient diagnostic tests and tools for epidemiological studies, and perhaps allow prediction and prevention of the acute reactionary episodes that can intensify nerve damage during the clinical course of the disease.