Marie Lipoldová

Genetic susceptibility to infectious disease: lessons from mouse models of leishmaniasis

Susceptibility to infectious disease is influenced by multiple host genes, most of which are low penetrance QTLs that are difficult to map in humans. Leishmaniasis is a well-studied infectious disease with a variety of symptoms and well-defined immunological features. Mouse models of this disease have revealed more than 20 QTLs as being susceptibility genes, studies of which have made important contributions to our understanding of the host response to infection. The functional effects of individual QTLs differ widely, indicating a networked regulation of these effects. Several of these QTLs probably also influence susceptibility to other infections, indicating that their characterization will contribute to our understanding of susceptibility to infectious disease in general.

Different Genetic Control of Cutaneous and Visceral Disease after Leishmania major Infection in Mice

ABSTRACT The mouse strains BALB/cHeA (BALB/c) and STS/A (STS) are susceptible and resistant to Leishmania major-induced disease, respectively. We analyzed this difference using recombinant congenic (RC) BALB/c-c-STS/Dem (CcS/Dem) strains that carry different random subsets of 12.5% genes of the strain STS in a BALB/c background. Previously, testing the resistant strain CcS-5, we found five novel Lmr (Leishmania major response) loci, each associated with a different combination of pathological and immunological reactions. Here we analyze the response of RC strain CcS-16, which is even more susceptible to L. major than BALB/c. In the (CcS-16 × BALB/c)F2 hybrids we mapped three novel loci that influence cutaneous or visceral pathology. Lmr14 (chromosome 2) controls splenomegaly and hepatomegaly. On the other hand Lmr15 (chromosome 11) determines hepatomegaly only, and Lmr13 (chromosome 18) determines skin lesions only. These data confirm the complex control of L. major-induced pathology, where cutaneous and visceral pathology are controlled by different combinations of genes. It indicates organ-specific control of antiparasite responses. The definition of genes controlling these responses will permit a better understanding of pathways and genetic diversity underlying the different disease phenotypes.

Mice with different susceptibility to tick-borne encephalitis virus infection show selective neutralizing antibody response and inflammatory reaction in the central nervous system

BackgroundThe clinical course of tick-borne encephalitis (TBE), a disease caused by TBE virus, ranges from asymptomatic or mild influenza-like infection to severe debilitating encephalitis or encephalomyelitis. Despite the medical importance of this disease, some crucial steps in the development of encephalitis remain poorly understood. In particular, the basis of the disease severity is largely unknown.MethodsTBE virus growth, neutralizing antibody response, key cytokine and chemokine mRNA production and changes in mRNA levels of cell surface markers of immunocompetent cells in brain were measured in mice with different susceptibilities to TBE virus infection.ResultsAn animal model of TBE based on BALB/c-c-STS/A (CcS/Dem) recombinant congenic mouse strains showing different severities of the infection in relation to the host genetic background was developed. After subcutaneous inoculation of TBE virus, BALB/c mice showed medium susceptibility to the infection, STS mice were resistant, and CcS-11 mice were highly susceptible. The resistant STS mice showed lower and delayed viremia, lower virus production in the brain and low cytokine/chemokine mRNA production, but had a strong neutralizing antibody response. The most sensitive strain (CcS-11) failed in production of neutralizing antibodies, but exhibited strong cytokine/chemokine mRNA production in the brain. After intracerebral inoculation, all mouse strains were sensitive to the infection and had similar virus production in the brain, but STS mice survived significantly longer than CcS-11 mice. These two strains also differed in the expression of key cytokines/chemokines, particularly interferon gamma-induced protein 10 (IP-10/CXCL10) and monocyte chemotactic protein-1 (MCP-1/CCL2) in the brain.ConclusionsOur data indicate that the genetic control is an important factor influencing the clinical course of TBE. High neutralizing antibody response might be crucial for preventing host fatality, but high expression of various cytokines/chemokines during TBE can mediate immunopathology and be associated with more severe course of the infection and increased fatality.

The protective effect against Leishmania infection conferred by sand fly bites is limited to short-term exposure

Under laboratory conditions, hosts exposed twice to sand fly saliva are protected against severe leishmaniasis. However, people in endemic areas are exposed to the vector over a long term and may experience sand fly-free periods. Therefore, we exposed mice long- or short-term to Phlebotomus duboscqi bites, followed by Leishmania major infection either immediately or after a sand fly-free period. We showed that protection against leishmaniasis is limited to short-term exposure to sand flies immediately before infection. Our results may explain the persistence of leishmaniasis in endemic areas and should be taken into account when designing anti-Leishmania vaccines based on sand fly saliva.

Separation of multiple genes controlling the T-cell proliferative response to IL-2 and anti-CD3 using recombinant congenic strains.

T lymphocytes of the strain BALB/cHeA exhibit a low proliferative response to IL-2 and a high response to the anti-CD3 monoclonal antibodies, while the strain STS/A lymphocyte response to these stimuli is the opposite. We analyzed the genetic basis of this strain difference, using a novel genetic tool: the recombinant congenic strains (RCS). Twenty BALB/c-c-STS/Dem (CcS/Dem) RCS were used, each containing a different random set of approximately 12.5% of the genes from STS and the remainder from BALB/c. Consequently, the genes participating in the multigenic control of a phenotypic difference between BALB/c and STS become separated into different CcS strains where they can be studied individually. The strain distribution patterns of the proliferative responses to IL-2 and anti-CD3 in the CcS strains are different, showing that different genes are involved. The large differences between individual CcS strains in response to IL-2 or anti-CD3 indicate that both reactions are controlled by a limited number of genes with a relatively large effect. The high proliferative response to IL-2 is a dominant characteristic. It is not caused by a larger major cell subset size, nor by a higher level of IL-2R expression. The response to anti-CD3 is known to be controlled by polymorphism in Fcγ receptor 2 (Fcgr2) and the CcS strains carrying the low responder Fcgr2 allele indeed responded weakly. However, as these strains do respond to immobilized anti-CD3, while the STS strain does not, and as some CcS strains with the BALB/c allele of Fcgr2 are also low responders, additional gene(s) of the STS strain strongly depress the anti-CD3 response. In a backcross between the high responder and the low responder strains CcS-9 and CcS-11, one of these unknown genes was mapped to the chromosome 10 near D10Mit14. The CcS mouse strains which carry the STS alleles of genes controlling the proliferative response to IL-2 and anti-CD3 allow the future mapping, cloning, and functional analysis of these genes and the study of their biological effects in vivo.

Mouse genetic model for clinical and immunological heterogeneity of leishmaniasis

Abstract. Systematic assessment of the role of host genes in clinico-pathological and immunological manisfestations of Leishmania major-induced disease in mice was performed using 20 recombinant congenic (RC) strains. As the RC strains are homozygous and each carries a different, random set of 12.5% genes from the resistant strain, STS/A, and 87.5% genes from the susceptible strain, BALB/cHeA, they allowed us to study the pathological and immunological characteristics of infected hosts in 20 fixed different random combinations of BALB/c and STS genes. The 20 RC strains differ widely in expression of different symptoms of disease and in immunological characteristics. Disease or healing in different strains occurred in association with different components of immune response – with the exception of a frequently occurring correlation between the disease and IgE levels. 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. Our data also suggest that despite the complexity of regulation, when a sufficient number of controlling loci is known, the prediction of a phenotype is possible. Combining functional and clinical information with multilocus genotyping may improve our ability to predict the progression of the disease and to optimize the treatment.

Leishmania parasite detection and quantification using PCR-ELISA

This protocol describes an improved and optimized PCR-ELISA method for detection and quantification of Leishmania parasites in host tissues. Unlike other DNA-based assays, this method uses digoxigenin- and biotin-labeled primers. This eliminates the need for a separate step of hybridization of the PCR product with labeled probes. The PCR product is detected using sandwich ELISA with antidigoxigenin-detecting antibodies. Primers are complementary to the kinetoplast minicircle conserved region of parasite DNA, allowing the detection of several Leishmania species. For measurement of a wide range of parasite concentrations, ±25 cycles were optimal. The sensitivity of this technique is 0.3 fg of parasite DNA per reaction in 40-cycle PCR-ELISA, corresponding to 0.004 parasites. DNA preparation by a standard TRI reagent procedure takes about 4 h. When DNA is prepared, a single person can test a large number of samples (at least 150) in a maximum of 7 h. This method might also be suitable for detecting and quantifying other pathogens, especially for detecting small differences in pathogen numbers.

Distinct genetic control of parasite elimination, dissemination, and disease after Leishmania major infection

Elimination of pathogens is the basis of host resistance to infections; however, relationship between persisting pathogens and disease has not been clarified. Leishmania major infection in mice is an important model of host–pathogen relationship. Infected BALB/c mice exhibit high parasite numbers in lymph nodes and spleens, and a chronic disease with skin lesions, splenomegaly, and hepatomegaly, increased serum IgE levels and cytokine imbalance. Although numerous gene loci affecting these disease symptoms have been reported, genes controlling parasites’ elimination or dissemination have never been mapped. We therefore compared genetics of the clinical and immunologic symptomatology with parasite load in (BALB/c × CcS-11) F2 hybrids and mapped five loci, two of which control parasite elimination or dissemination. Lmr5 influences parasite loads in spleens (and skin lesions, splenomegaly, and serum IgE, IL-4, and IFNγ levels), and Lmr20 determines parasite numbers in draining lymph nodes (and serum levels of IgE and IFNγ), but no skin or visceral pathology. Three additional loci do not affect parasite numbers but influence significantly the disease phenotype—Lmr21: skin lesions and IFNγ levels, Lmr22: IL-4 levels, Lmr23: IFNγ levels, indicating that development of L. major-caused disease includes critical regulations additional to control of parasite spread.

Modulation of murine cellular immune response and cytokine production by salivary gland lysate of three sand fly species

Saliva of sand flies ( Diptera: Phlebotominae ) plays an important role in transmission of Leishmania parasites by modulating host immune response. However, because of the different protein compositions of saliva, the immunomodulatory effects may vary among sand fly species. We have therefore analysed and compared the immunomodulation effects of salivary gland lysate (SGL) of three different sand flies. Spleen cells from BALB/c mice were incubated with SGL of Phlebotomus papatasi, P. sergenti or Lutzomyia longipalpis. Concanavalin A‐stimulated lymphocyte proliferation was significantly suppressed with SGLs of all three sand fly species and all SGL doses tested. This result indicates that saliva from different sand fly species is able to suppress host proliferative response even to the potent mitogen. In parallel experiments, we analysed the effect of SGL on IFN‐γ, IL‐2, and IL‐4 production; in mitogen‐stimulated cells SGLs markedly inhibited IFN‐γ production in all intervals tested (reduced up to 31%) and to a lesser degree impaired production of the other two cytokines as well. Despite some species‐specific differences in the intensity of immunomodulatory effects, saliva of all sand fly species modulated cell proliferation as well as cytokine production in a similar way.

Preparation of highly infective Leishmania promastigotes by cultivation on SNB-9 biphasic medium

Protozoan hemoflagellates Leishmania are causative agents of leishmaniases and an important biological model for study of host-pathogen interaction. A wide range of methods of Leishmania cultivation on both biphasic and liquid media is available. Biphasic media are considered to be superior for initial isolation of the parasites and obtaining high promastigote infectivity; however, liquid media are more suitable for large-scale experiments. The aim of the present study was the adaptation and optimization of the cultivation of Leishmania promastigotes on a biphasic SNB-9 (saline-neopeptone-blood 9) medium that was originally developed for Trypanosoma cultivation and combines the advantages of biphasic and liquid media. SNB-9 medium is characterized with a large volume of the liquid phase, which facilitates the manipulation with the culture and provides parasite yields comparable to parasite yields on such liquid medium as Schneiders Insect Medium. We demonstrate that SNB-9 very considerably surpasses Schneiders Insect Medium in in vitro infectivity of the parasites. Additionally, we show that the ratio of apoptotic parasites, which are important for the infectivity of the inoculum, in Leishmania culture in SNB-9 is higher than in Leishmania culture in Schneiders Insect Medium. Thus, we demonstrate that the cultivation of Leishmania on SNB-9 reliably yields highly infective promastigotes suitable for experimental infection.