Research since 1995
The research program of the laboratory aims to identify genes and molecular mechanisms involved in control of immune response and susceptibility to complex infectious diseases. We focus on complex diseases because they are responsible for the largest part of human morbidity and mortality and their genetic analysis is subject of an intensive international effort. They are controlled by multiple genes and hence their pathogenesis cannot be explained by effects of a single gene with omission of others. Leishmaniasis is such a complex disease and it has served as a major paradigm of immune response to an infectious agent. We aim to identify the genes and functions controlling this disease.
We apply a special tool for genetic analysis of multigenically-controlled biological traits: the Recombinant Congenic Strains (RCS). The series of RCS comprises 20 homozygous mouse strains. Each RC strain contains a different, random, set of about 12.5% genes from a parental donor strain and about 87.5% genes of a parental background strain. In this way, the individual genes of the donor strain participating in the complex control of multigenically-controlled biological traits become divided into different RC strains, where they can be studied one by one. Thus, the RCS system transforms a multigenic difference into a set of a single gene differences (or oligogenic differences).
We have shown that the susceptibility to Leishmania major is multigenically controlled (Demant et al. 1996) and we have performed a systematic assessment of the role of host genes in clinico-pathological and immunological manifestations of L. major induced disease using 20 RC strains derived from the susceptible strain BALB/c and the resistant strain STS.
Disease or healing in different RC strains occurred in association with different components of immune response. Moreover, some parameters of the immune response were highly correlated in some strains but not at all in others. This shows that several patterns of the immune response may be associated with the same clinical outcome, depending on the host genotype (Lipoldova et al. 2002).
Definition of 21 novel Leishmania major response (Lmr) loci
The strains CcS-5, CcS-11, CcS-16 and CcS-20 were selected for genetic analysis because they exhibit diverse symptomatology after infection. The high resolving power of the RC system revealed 21 novel QTL (quantitative trait loci): Lmr3-Lmr23 (partly reviewed in Lipoldova and Demant 2006 and ref. Lipoldova et al. 2000, Badalova et al. 2002, Vladimirov et al. 2003, Havelkova et al. 2006a, Kurey et al. 2009). They included genes controlling skin lesions, hepatomegaly, splenomegaly, serum levels of IgE, IL-4, IL-6, IL-12, TNFα and IFNγ, and spontaneous proliferation of lymphocytes in infected mice. We developed a precise quantification method for Leishmania parasites (Kobets et al. 2010), which revealed for the first time the genes controlling parasite numbers and demonstrated their distinctness from susceptibility genes (Kurey et al. 2009). This provided a comprehensive insight into genetics of the multifaceted response to L. major infection.
Functional heterogeneity of Lmr loci and their regulatory networks
Effects of 21 Lmr genes on disease symptoms were organ specific and heterogeneous. The individual Lmr loci control 17 different combinations of pathological and immunological symptoms. Eight loci control both organ pathology and immunological parameters and 13 only immune reactions. 15 Lmr loci are involved in one or more genetic interactions showing that gene interactions are common in response to L. major. Moreover, parasite elimination, immunological and pathological processes are regulated partly independently (Kurey et al. 2009). In conclusion, these studies revealed a network-like complexity of the combined effects of the multiple functionally diverse QTLs (partly reviewed in Lipoldova and Demant 2006 and Lipoldova et al. 2000, Havelkova et al. 2006a, Kurey et al. 2009).
Possible relevance of Lmr loci for other diseases
Some Lmr loci co-localize with QTLs that influence response to other infectious agents, including both bacteria and parasites (Havelkova et al. 2006a, Lipoldova and Demant, 2006, Kurey et al. 2009). This suggests the presence either of clusters of functionally related genes, or of genes that are involved in controlling the response to several infections.
The hypothesis that Lmr loci are likely relevant also for other diseases was supported by our detection of a novel human atopy controlling locus at 8q12 in the position homologous to Lmr9 (Gusareva et al. 2009a).
Immunoglobulin E (IgE) plays an important role in response to parasites and in development of atopic reactions. The susceptible strain BALB/c exhibits a high, whereas a resistant strain a low serum IgE level after L. major infection ( Badalova et al. 2002 ).
We have analyzed the genetic basis of this strain differences using the low IgE producing strains CcS-5 and CcS-20, intermediate strain CcS-11 and a high IgE producing strain CcS-16. These RC strains cover approximately 40% differences between BALB/c and STS. These differences are controlled by ten Leishmania major response (Lmr) loci (Lipoldova et al. 2000, Badalova et al. 2002, Kurey et al. 2009). Despite the overall genetic similarity between the strains CcS-5, CcS-11, CcS-16 and CcS-20 (each has 87.5% of the BALB/c genome), only two loci control IgE level in more than one strain. This findings resembles the situation observed in studies of human atopy where linkage observed in one population in not seen in the other population. RC strains might therefore serve as a model of these different human families.
The human segments that were homologous to Lmr3, Lmr5, Lmr8, Lmr10, Lmr11, Lmr12, Lmr13, Lmr14 and Lmr20 in mouse had been already described in genome-wide scans for atopy and asthma loci in humans (partly reviewed in Gusareva et al. 2009a) However, one chromosomal segment identified from the homology with mouse (Lmr9) (Badalova et al. 2002) has not shown evidence for linkage with IgE or some allergic disorder in the previous studies in humans. A human region homologous to the Lmr9, which is located at a chromosomal segment 8q12 showed a suggestive linkage to the composite inhalant allergic sensitization and to nine specific IgEs (Gusareva et al. 2009a). This finding illustrates the efficiency and power of the genome-wide screening in mice in identification of loci/genes of complex traits such as IgE in humans.
Major allergens and prevalence in sensitization in atopic patients vary in different populations, providing cues about the pathogenic effects of environment and lifestyle. We estimated sensitization to 20 different airborne allergens in children and adolescent patients with atopic bronchial asthma from Tomsk and Thumen, cities of the west Siberia, Russia (map). Cat (e1) was found to be the major allergen with 57.3% of Russian asthmatic patients sensitized to this allergen. Other important allergens were D. pteronyssinus (d1), D. farinae (d2) and dog allergen (e2), with more than 30% of asthmatics sensitized. This is the first report about the distribution of specific IgE to airborne allergens in the asthmatic patients from the Russia (Gusareva et al. 2006) .
Locus controlling cat-specific IgE in asthma patients from Siberian population was mapped within the interval between 136 and 140 cM on chromosome 12q24.3 (Gusareva et al. 2009b).
Many biological processes and diseases, including cancer and cardiovascular diseases are in their onset driven by non-immunological mechanisms, but their further development may be strongly modified by immunological, particularly inflammatory responses. In our research we have separated (Lipoldova et al. 1995, Holan et al. 1996, Havelkova et al. 2006b) and mapped 21 novel loci that control T cell proliferative response to IL-2 (Krulova et al. 1997; Lipoldova et al. 2005 ), IL-4 (Lipoldova et al. 2005 ), anti-CD3 (Havelkova et al. 1996, 1999a, 1999b), and to alloantigens (Holan et al. 2000; Havelkova et al. 2000), and loci influencing production of cytokines (Kosarova et al. 1999, Lipoldova et al. 2010). These data were used to establish a genetic links between loci controlling activation of lymphocytes and cancer suceptibility loci.
Low infiltration of lymphocytes into cancers is associated with poor prognosis, but the reasons why some patients exhibit a low and others a high infiltration of tumors are unknown. Previously were mapped four loci (Lynf1 – Lynf4) controlling lymphocyte infiltration of mouse lung tumors. We have found a genetic relationship between these loci and the control of production of IFNg in allogeneic mixed lymphocyte cultures influenced by loci Cypr4 (Cytokine production 4)-Cypr7. This suggests that inherited differences in certain lymphocyte responses may modify their propensity to infiltrate tumors and their capacity to affect tumor progression (Lipoldova et al. 2010).
Similarly, all loci, which we found to control T cell proliferation (Cinda1 – 5, Tria1-5, Alan1,2), co-localized with loci controlling cancer susceptibility and several of them also with loci controlling macrophage activation and autoimmunity. Four from the five Cinda loci that control cytokine-induced activation have been surprisingly found to be associated with the lung cancer susceptibility (Krulova et al. 1997, Lipoldova et al. 2005). This suggests that the differences in tumor susceptibility attributed to the Sluc loci may actually be due to differences in lymphocyte-cytokine interactions and that Sluc loci merely reflect the impact of Cinda genes on tumorigenesis. Alternatively, Cinda and Sluc genes are distinct and occur in several closely linked complexes in the genome. The involvement of immune functions in Sluc effects is supported by the observation that independently detected susceptibility loci for proteoglycan-induced arthritis apparently map within 1 cM of three out of the four Sluc loci that co-localize with Cinda loci: Cinda4-Sluc1 - Pgia12, Cinda1-Sluc4 - Pgia7, and Cinda5-Sluc20 - Pgia4 (figure). The possible basis of relationship between genetic control of cytokine responsiveness and tumour susceptibility may be also due to common signalling pathways stimulated by IL-2 and/or IL-4 in lymphocytes and in tumor cells. Identification of Cinda and Sluc genes will likely reveal important novel aspects of both lymphocyte function and tumorigenesis.
The finding that most of Cypr, Cinda, Tria and Alan loci are linked with genes controlling cancer susceptibility suggests that genetic control of immune responses may be responsible for a considerable part of genetic differences in tumor susceptibility.