Ingrid M ¨uller 1 and Pascale Kropf 2
7.5 Adaptive immunity: lessons from L. major infections of mice
In all forms of leishmaniasis, both immunity and pathology are mediated predominantly by T lymphocytes. Experimental studies in inbred strains of mice with L. major have contributed significantly to our understanding of host/parasite interactions, as well as establishing basic immunological princi- ples such as the involvement of the Th1/Th2 paradigm of T helper (Th) cell sub- sets in infectious diseases. Therefore, this chapter will now focus on immune responses toL. majorinfections in mice.
Inbred strains of mice are usually infected by needle inoculation withL. major promastigotes (doses of 104–107). This inoculation can be performed at differ- ent sites, but the most commonly used are the hind footpads, the rump, or the base of the tail. Of note, there are different ways to perform infection of mice withLeishmaniaparasites, and this can result in different outcomes of infec- tion. Factors which impact on the immune response generated include injec- tion of lower numbers of parasites, different developmental stages of parasites and the route and site of injection in the mouse.
7.5.1 CD4+ T cells
In the classical model of infection of mice withL. major, control of infection and healing have been associated with a polarised Th1 response, whereas non- healing has been ascribed to an interleukin (IL)-4-dominated polarised Th2 re- sponse (Figure 7.2).
The majority of inbred strains of mice, such as CBA or C57BL/6, develop small lesions that will spontaneously heal within a few weeks, leaving the mice im- mune to reinfection. This ability to control infection is associated with the pref- erential expansion of antigen-specific CD4+ Th1 cells, characterised by the pro- duction of interferon (IFN)-␥. Indeed, mice from a genetically resistant back- ground lacking the IFN-␥receptor cannot controlL. majorinfection.
Th1 cells efficiently contribute to the control of parasites by promoting the abil- ity of macrophages to kill the intracellularLeishmaniaparasites via the induc- tion of nitric oxide (NO), a key anti-microbial effector molecule (Figure 7.2).
DC C57BL/6 Healing / Resistant
CD4+
Th1 CD4+
CD4+ Th1
Th0 CD4+
Th1 IFN-γ IL-12
DC BALB/c Non-healing / Susceptible
CD4+
CD4+ Th2 Th0
CD4+
Th2 CD4+
Th2
IL-4 IL-5 IL-13
Classically activated macrophages (M1) NO-mediated death of
Leishmania
Alternatively activated macrophages (M2) Arginase-mediated hydrolysis of L-arginine to
L-ornithine, a polyamine used for parasite growth.
Figure 7.2 Resistance and susceptibility toLeishmaniain mouse models: the Th1/Th2 paradigm.
NO is generated from L-arginine by the enzyme inducible NO synthase (iNOS), and iNOS-deficient mice are unable to controlL. majorinfections, even though their capacity to mount a strong Th1 response is not impaired. Polarisation of Th1 cells is mainly directed by the production of IL-12 by dendritic cells (DC).
Early studies have shown that infection of resistant mice withL. majorinduces IL-12 productionin vivo, and that IL-12 is essential for the development of pro- tective Th1 response. Further, uptake ofL. majorby dendritic cells leads to the production of IL-12 and the subsequent priming of Th1 cells. The selective loss of IL-12 signalling by antigen-specific CD4+ T cells has been proposed to con- tribute to the susceptibility of BALB/c mice toL. majorinfection.
In contrast to the healing phenotype, a few strains of mice, such as BALB/c, develop non-healing disease following infection withL. majorparasites. This non-healing phenotype is attributed to the preferential expansion of Th2 cells, characterised by the production of IL-4, IL-5 and IL-13. The development of a Th2 response in BALB/c mice infected by L. majorhas been attributed to the early production of IL-4 by V4V␣8 CD4+ T cells. These cells are known to recognise theLeishmaniaantigen LACK (Leishmaniahomolog of receptors for activated C kinase), and they can be detected as early as 16 hours post in- fection. LACK-specific CD4+ T cells are known to be involved in Th2-mediated susceptibility toL. majorinfection, because mice made tolerant to LACK dis- play a decreased Th2 response and controlL. majorinfection. In addition, V4- deficient BALB/c mice display a stronger Th1 response than genetically intact BALB/c mice and are able to control parasite replication.
IL-4 is important in the development of Th2 responses and the associated non- healing phenotype of BALB/c mice following infection withL. majorparasites, and treatment of BALB/c mice with anti-IL-4 monoclonal antibodies (mAb) re- sults in control of parasite replication. However, despite these results, there is now mounting evidence that IL-4 is not always sufficient, or even necessary, for susceptibility. Even in the face of a protective Th1 response, non-healing lesions can develop in a genetically resistant strain of mice infected with a virulent strain ofL. majorin the absence of IL-4. Furthermore, IL-4-deficient BALB/c mice infected withL. majorsub-strains IR173 or LV39 do not generate a pro- tective Th1 response and are only slightly less susceptible, or as susceptible, as wild-type mice, indicating that IL-4 is not essential for non-healing.
The reason why IL-4 is not necessary for susceptibility is because, in mice, a non-healing phenotype is multi-factorial. The IL-4 receptor␣-chain (IL-4R␣) is a component of both the IL-4 receptor and the IL-13 receptor; IL-4R␣-deficient mice can control infection with L. major IR173, suggesting that IL-13 plays a role in susceptibility toL. major infection. Indeed, experiments performed in IL-13 deficient mice confirmed the importance of IL-13 as a susceptibility factor.
Susceptibility can also be mediated by the suppression of protective Th1 re- sponses by the immunomodulatory cytokine IL-10. This cytokine can be pro- duced by many cell types, including B cells, macrophages, DCs and some pop- ulations of T cells, such as regulatory T cells (Treg).L. major-infected BALB/c mice that are genetically deficient in IL-10 are more resistant to infection than
their wild-type counterparts, and blocking the IL-10 receptor with a mAb re- sults in more efficient control of parasite replication.
Recently, a novel population of Leishmania-specific T cells that are CD4+CD25−Foxp3− produce both IL-10 and IFN-␥ have been shown to be immunosuppressive and abrogate acquired immunity. This T cell popu- lation may be an important source of IL-10, and their development during Leishmaniainfection may play an important role in mediating a non-healing susceptible phenotype.
7.5.2 Regulatory T cells
Tregs are essential for the maintenance of immunological homeostasis. Sev- eral types of regulatory T cells have been described to date, some of which develop during selection in the thymus (natural Tregs), and some of which are induced in response to infectious challenge (induced Tregs). Natural Tregs play an important role in the regulation of the immune response to Leish- maniaparasites. These cells, constituting 5–10 per cent of peripheral CD4+ T cells (both in mice and humans), have been shown to control excessive im- mune responses. The hallmarks of natural Tregs are the surface expression of the IL-2R␣-chain (CD25) and the intracellular expression of the transcription factor FoxP3. However, the expression of molecules such as Cytotoxic T lym- phocyte antigen (CTLA)-4 and glucocorticoid-induced TNFR-related protein (GITR) have also been associated with these cells.
When genetically resistant mouse strains heal,Leishmaniaparasites persist in the body (latency). Pioneer work with the low-dose intradermal challenge in a genetically resistant strain of mice has shown that natural Tregs accumulate at the site of inoculation, where they suppress the ability of CD4+ T effector cells to eliminateLeishmaniaparasites once pathogenesis has been resolved. Natu- ral Tregs can use both IL-10-dependent and IL-10-independent mechanisms to contribute to parasite persistence.
Interestingly, in this study, when parasites were eliminated and sterile cure was achieved, the capacity of cured mice to control secondary infections was ab- rogated, demonstrating that equilibrium between Tregs and effector T cells is crucial for immunity to reinfection. This study also showed that natural Tregs proliferate in response toL. major-infected DCs, further supporting the con- cept that natural Treg can recognise microbial antigens.
7.5.3 Th17 cells
Recently a new subset of T helper cells, Th17, has been described; these cells produce the pro-inflammatory cytokine, IL-17. Macrophages, endothelial cells and fibroblasts produce inflammatory mediators such as TNF-␣, IL-1 and chemokines in response to IL-17 which, in turn, results in the recruitment of neutrophils and leukocytes. Th17 cells have been shown to play an impor- tant role in inflammatory diseases, and a recent study has shown that IL-17 deficient mice on a susceptible background were able to controlLeishmania
parasite load more efficiently than intact mice. IL-17 did not seem to play a role in the differentiation of Th1 and Th2 immune responses in this study. Rather, a reduction of neutrophil recruitment to the site of infection reduced the initial survival ofLeishmaniaparasites (presumably by reducing the successful entry of parasites into macrophages).
7.5.4 CD8+ T cells
No protective role for CD8+ T cells has been shown so far in the control of pri- maryLeishmaniainfections in the standard mouse model of cutaneous leish- maniasis, i.e. subcutaneous infection with high dose of parasites. Mice deficient in CD8+ T cells or in MHC-I (and therefore unable to present antigen to CD8+
T cells) remain resistant toLeishmaniainfection. In contrast, in low-dose mod- els of intradermal infection with L. majorparasites, CD8+ T cells participate in the pathogenesis and immunity to primary infection. Genetically resistant mice deficient in CD8+ T cells fail to control infection under these conditions.
CD8+ T cells undergo expansion and produce IFN-␥ in genetically resistant immunologically intact mice injected intradermally with low doses ofL. ma- jor. Primed CD8+ T cells from these mice can transfer protective immunity to recombination-activating gene (RAG)-deficient mice (these mice lack both T and B cells) infected withL. major.Therefore, the role of CD8+ T cells in immu- nity toLeishmaniaappears to depend on the dose of parasites administered, and this may be highly relevant in humans, whereby infection from the bite of a sandfly arises from a low dose ofLeishmaniaparasites.