Effects of Leishmania major infection on the gut microbiome of resistant and susceptible mice.

Cutaneous leishmaniasis, a parasitic disease caused by Leishmania major, is a widely frequent form in humans. To explore the importance of the host gut microbiota and to investigate its changes during L. major infection, two different groups of mouse models were assessed. The microbiome of two parts of the host gut-ileum and colon-from infected and non-infected mice were characterised by sequencing of 16S rDNA using an Ion Torrent PGM platform. Microbiome analysis was performed to reveal changes related to the susceptibility and the genetics of mice strains in two different gut compartments and to compare the results between infected and non-infected mice. The results showed that Leishmania infection affects mainly the ileum microbiota, whereas the colon bacterial community was more stable. Different biomarkers were determined in the gut microbiota of infected resistant mice and infected susceptible mice using LEfSe analysis. Lactobacillaceae was associated with resistance in the colon microbiota of all resistant mice strains infected with L. major. Genes related to xenobiotic biodegradation and metabolism and amino acid metabolism were primarily enriched in the small intestine microbiome of resistant strains, while genes associated with carbohydrate metabolism and glycan biosynthesis and metabolism were most abundant in the gut microbiome of the infected susceptible mice. These results should improve our understanding of host-parasite interaction and provide important insights into the effect of leishmaniasis on the gut microbiota. Also, this study highlights the role of host genetic variation in shaping the diversity and composition of the gut microbiome. KEY POINTS: • Leishmaniasis may affect mainly the ileum microbiota while colon microbiota was more stable. • Biomarkers related with resistance or susceptibility were determined in the gut microbiota of mice. • Several pathways were predicted to be upregulated in the gut microbiota of resistant or susceptible mice.

Functionally distinct regions of the locus Leishmania major response 15 control IgE or IFNγ level in addition to skin lesions.

Leishmaniasis, a disease caused by parasites of Leishmania spp., endangers more than 1 billion people living in endemic countries and has three clinical forms: cutaneous, mucocutaneous, and visceral. Understanding of individual differences in susceptibility to infection and heterogeneity of its pathology is largely lacking. Different mouse strains show a broad and heterogeneous range of disease manifestations such as skin lesions, splenomegaly, hepatomegaly, and increased serum levels of immunoglobulin E and several cytokines. Genome-wide mapping of these strain differences detected more than 30 quantitative trait loci (QTLs) that control the response to Leishmania major. Some control different combinations of disease manifestations, but the nature of this heterogeneity is not yet clear. In this study, we analyzed the L. major response locus Lmr15 originally mapped in the strain CcS-9 which carries 12.5% of the genome of the resistant strain STS on the genetic background of the susceptible strain BALB/c. For this analysis, we used the advanced intercross line K3FV between the strains BALB/c and STS. We confirmed the previously detected loci Lmr15, Lmr18, Lmr24, and Lmr27 and performed genetic dissection of the effects of Lmr15 on chromosome 11. We prepared the interval-specific recombinant strains 6232HS1 and 6229FUD, carrying two STS-derived segments comprising the peak linkage of Lmr15 whose lengths were 6.32 and 17.4 Mbp, respectively, and analyzed their response to L. major infection. These experiments revealed at least two linked but functionally distinct chromosomal regions controlling IFNγ response and IgE response, respectively, in addition to the control of skin lesions. Bioinformatics and expression analysis identified the potential candidate gene Top3a. This finding further clarifies the genetic organization of factors relevant to understanding the differences in the individual risk of disease.

Genetic Influence on Frequencies of Myeloid-Derived Cell Subpopulations in Mouse.

Differences in frequencies of blood cell subpopulations were reported to influence the course of infections, atopic and autoimmune diseases, and cancer. We have discovered a unique mouse strain B10.O20 containing extremely high frequency of myeloid-derived cells (MDC) in spleen. B10.O20 carries 3.6% of genes of the strain O20 on the C57BL/10 genetic background. It contains much higher frequency of CD11b+Gr1+ cells in spleen than both its parents. B10.O20 carries O20-derived segments on chromosomes 1, 15, 17, and 18. Their linkage with frequencies of blood cell subpopulations in spleen was tested in F2 hybrids between B10.O20 and C57BL/10. We found 3 novel loci controlling MDC frequencies: Mydc1, 2, and 3 on chromosomes 1, 15, and 17, respectively, and a locus controlling relative spleen weight (Rsw1) that co-localizes with Mydc3 and also influences proportion of white and red pulp in spleen. Mydc1 controls numbers of CD11b+Gr1+ cells. Interaction of Mydc2 and Mydc3 regulates frequency of CD11b+Gr1+ cells and neutrophils (Gr1+Siglec-F- cells from CD11b+ cells). Interestingly, Mydc3/Rsw1 is orthologous with human segment 6q21 that was shown previously to determine counts of white blood cells. Bioinformatics analysis of genomic sequence of the chromosomal segments bearing these loci revealed polymorphisms between O20 and C57BL/10 that change RNA stability and genes' functions, and we examined expression of relevant genes. This identified potential candidate genes Smap1, Vps52, Tnxb, and Rab44. Definition of genetic control of MDC can help to personalize therapy of diseases influenced by these cells.

Role of host genetics and cytokines in Leishmania infection.

Cytokines and chemokines are important regulators of innate and specific responses in leishmaniasis, a disease that currently affects 12 million people. We overviewed the current information about influences of genetically engineered mouse models of cytokine and chemokine on leishmaniasis. We found that genetic background of the host, parasite species and sub-strain, as well as experimental design often modify effects of genetically engineered cytokine genes. Next we analyzed genes and QTLs (quantitative trait loci) that control response to Leishmania species in mouse in order to establish relationship between genetic control of cytokine expression and organ pathology. These studies revealed a network-like complexity of the combined effects of the multiple functionally diverse QTLs and their individual specificity. Genetic control of organ pathology and systemic immune response overlap only partially. Some QTLs control both organ pathology and systemic immune response, but the effects of genes and loci with the strongest impact on disease are cytokine-independent, whereas several loci modify cytokines levels in serum without influencing organ pathology. Understanding this genetic control might be important in development of vaccines designed to stimulate certain cytokine spectrum.

In vitro effects of azide-containing human CRP isoforms and oxLDL on U937-derived macrophage production of atherosclerosis-related cytokines.

Atherosclerosis is an inflammatory chronic disease of the arterial wall. Monomeric (m) and pentameric (p) C-reactive protein (CRP) and oxidized low density lipoproteins (oxLDL) seem to affect the pattern of cytokine production by macrophages, thus playing an important role in atherogenesis. Azide, the commercial preservative of CRP, may influence its action in vitro. The present study aimed to determine the effects of both isoforms of azide-containing CRP (mCRP and pCRP) with and without oxLDL on cytokine production by U937-derived macrophages. U937 monocytes were cultured and differentiated into macrophages and treated with mCRP, pCRP, oxLDL and azide individually and in combination. ELISA were performed to measure the levels of interferon-γ (IFN-γ), interleukin (IL)-4, IL-6, IL-10 and tumor necrosis factor (TNF)-α in culture supernatants collected from U937-derived macrophages following their respective treatments. Most single and combined treatments, especially in triple combination, were able to downregulate the levels of IFN-γ and IL-6 compared with control untreated cells, whilst the combination of mCRP and pCRP increased IL-4 levels. Regarding IL-10, except for an increase induced by mCRP, no significant effect was caused by any treatment compared with the control. On the other hand, the levels of TNF-α were not significantly affected by any treatment except for a decreasing trend that was observed with mCRP/oxLDL treatment compared with control. By contrast, double azide caused a significant decrease in the levels of IFN-γ and IL-6. The results of the present study indicated that mCRP, pCRP, oxLD and possibly azide, individually or in different combinations, had the tendency to upregulate the expression of IL-4 and to downregulate that of the pro-atherogenic cytokines, IFN-γ and IL-6, suggesting that the intima microenvironment serves a crucial role in atherogenesis.

Genetic Regulation of Guanylate-Binding Proteins 2b and 5 during Leishmaniasis in Mice.

Interferon-induced GTPases [guanylate-binding proteins (GBPs)] play an important role in inflammasome activation and mediate innate resistance to many intracellular pathogens, but little is known about their role in leishmaniasis. We therefore studied expression of Gbp2b/Gbp1 and Gbp5 mRNA in skin, inguinal lymph nodes, spleen, and liver after Leishmania major infection and in uninfected controls. We used two different groups of related mouse strains: BALB/c, STS, and CcS-5, CcS-16, and CcS-20 that carry different combinations of BALB/c and STS genomes, and strains O20, C57BL/10 (B10) and B10.O20, OcB-9, and OcB-43 carrying different combinations of O20 and B10 genomes. The strains were classified on the basis of size and number of infection-induced skin lesions as highly susceptible (BALB/c, CcS-16), susceptible (B10.O20), intermediate (CcS-20), and resistant (STS, O20, B10, OcB-9, OcB-43). Some uninfected strains differed in expression of Gbp2b/Gbp1 and Gbp5, especially of Gbp2b/Gbp1 in skin. Uninfected BALB/c and STS did not differ in their expression, but in CcS-5, CcS-16, and CcS-20, which all carry BALB/c-derived Gbp gene-cluster, expression of Gbp2b/Gbp1 exceeds that of both parents. These data indicate trans-regulation of Gbps. Infection resulted in approximately 10× upregulation of Gbp2b/Gbp1 and Gbp5 mRNAs in organs of both susceptible and resistant strains, which was most pronounced in skin. CcS-20 expressed higher level of Gbp2b/Gbp1 than both parental strains in skin, whereas CcS-16 expressed higher level of Gbp2b/Gbp1 than both parental strains in skin and liver. This indicates a trans-regulation present in infected mice CcS-16 and CcS-20. Immunostaining of skin of five strains revealed in resistant and intermediate strains STS, CcS-5, O20, and CcS-20 tight co-localization of Gbp2b/Gbp1 protein with most L. major parasites, whereas in the highly susceptible strain, BALB/c most parasites did not associate with Gbp2b/Gbp1. In conclusion, expression of Gbp2b/Gbp1 and Gbp5 was increased even in organs of clinically asymptomatic resistant mice. It suggests a hidden inflammation, which might contribute to control of persisting parasites. This is supported by the co-localization of Gbpb2/Gbp1 protein and L. major parasites in skin of resistant and intermediate but not highly susceptible mice.

The combination of CRP isoforms with oxLDL decreases TNF-α and IL-6 release by U937-derived macrophages.

C-reactive protein (CRP) and oxidized low density lipoprotein (oxLDL) serve major roles at both early and advanced stages of atherosclerosis. CRP exists in two isoforms, monomeric (m) and pentameric (p), that bring about pro- or anti-inflammatory effects in macrophages. In addition, CRP may form a complex with oxidized low-density lipoprotein (oxLDL) via phosphatidylcholine, thus decreasing its pro-inflammatory effects within macrophages. The aim of the present study was to investigate the single and the combined effects of mCRP, pCRP and oxLDL on U937-derived macrophages. In the current study, U937-derived macrophages were treated in vitro with different combinations of CRP isoforms with or without oxLDL. The levels of major inflammatory cytokines [interleukin (IL)-1ß, IL-6, IL-8 and tumor necrosis factor (TNF)-α] along with the production of reactive oxygen species (ROS) were determined. TNF-α and IL-6 levels were significantly decreased (P<0.05) by the effect of mCRP and pCRP combined with oxLDL. No significant changes were observed in IL-1ß, IL-8 or ROS levels.