Nonhealing infection despite Th1 polarization produced by a strain of Leishmania major in C57BL/6 mice

Experimental Leishmania major infection in mice has been of immense interest because it was among the first models to demonstrate the importance of the Th1/Th2 balance to infection outcome in vivo. However, the Th2 polarization that promotes the development of nonhealing cutaneous lesions in BALB/c mice has failed to adequately explain the mechanisms underlying nonhealing forms of leishmaniasis in humans. We have studied a L. major strain from a patient with nonhealing lesions that also produces nonhealing lesions with ulcerations and high parasite burden in conventionally resistant C57BL/6 mice. Surprisingly, these mice develop a strong, polarized, and sustained Th1 response, as evidenced by high levels of IFN-gamma produced by Leishmania-specific cells in the draining lymph node and in the ear lesion, and an absence of IL-4 or IL-13. The parasites fail to be effectively cleared despite high level induction of inducible NO synthase in the lesion, and despite their sensitivity to killing by IFN-gamma-activated macrophages in vitro. Infection of IL-10(-/-) mice, blockade of the IL-10R, or depletion of CD25(+) cells during the chronic phase promotes parasite killing, indicating that IL-10 and regulatory T cells play a role in rendering the Th1 responses ineffective at controlling infection in the skin. Mice with nonhealing primary lesions are nonetheless resistant to reinfection in the other ear. We suggest that nonhealing infections in animal models that are explained not by aberrant Th2 development, but by overactivation of homeostatic pathways designed to control inflammation, provide better models to understand nonhealing or reactivation forms of leishmaniasis in humans.     

CD8+ T cells are required for primary immunity in C57BL/6 mice following low-dose, intradermal challenge with Leishmania major

Standard murine models of cutaneous leishmaniasis, involving s.c. inoculation of large numbers of Leishmania major promastigotes, have not supported an essential role for CD8(+) T cells in the control of primary infection. Recently, a L. major model combining two main features of natural transmission, low parasite dose and inoculation into a dermal site, has been established in resistant C57BL/6 mice. In the present studies, C57BL/6 mice with CD8(+) T cell deficiencies, including CD8(-/-) and CD8-depleted mice, failed to control the growth of L. major following inoculation of 100 metacyclic promastigotes into the ear dermis. The resulting dermal pathology was minor and delayed. Lesion formation in wild-type mice was coincident with the killing of parasites in the inoculation site. Both events were associated with the accumulation of CD8(+) T lymphocytes in the skin and with the capacity of CD8(+) T cells recovered from draining lymph nodes or infected dermis to release IFN-gamma following coculture with infected dendritic cells. Reconstitution of resistance to L. major in RAG(-/-) mice using T cells from naive donors was optimal when both CD4(+) and CD8(+) T cells were transferred. Primed CD8(+) T lymphocytes obtained from C57BL/6 mice during the acute stage of infection were able to mediate both pathology and immunity when transferred alone. The low dose, intradermal challenge model reveals that CD8(+) T cells play an essential role in both pathogenesis of and immunity to primary infection with L. major in the skin.     

Efficacy of the vaccination of C57BL/6 mice against infection with different species of Leishmania

Antigens were isolated from lysates of promastigotes of Leishmania infantum by electro-elution from polyacrylamide gels. Antigens with respective molecular weights for F2 = 94-67 kd; F5 = 30-20 kd and F6 below 20 kd, were injected intravenously in C 57 BL/6 mice. The immune sera were studied by indirect immunofluorescence; an in vivo test showed their inhibitory effect on the life cycle of several Leishmania species from the Old and the New world. Furthermore, mice immunized with F2, F5 or F6 were protected against an infection by Leishmania major. These results demonstrate that vaccination is efficient in mice differing genetically for either susceptibility (BALB/c) or partial resistance (C 57 BL/6) to Leishmania infections. Recently, we reported that a single monoclonal antibody raised against Leishmania infantum can prevent the development of Leishmania major and Leishmania mexicana amazonensis. Indeed, BALB/c mice injected subcutaneously with promastigotes pre-treated with this monoclonal antibody, did not present any cutaneous lesions over a period of 3 months. Using a mouse in vivo model--intraperitoneal injection of TG 180 mouse sarcoma cells along with monoclonal antibody pre-treated promastigotes--such an antibody-mediated inhibition was also observed against Old and New World Leishmania species. Protective monoclonal antibodies recognized by immunoblotting technique three antigenic fractions (40, 70 and 113 kd) common to several Leishmania species. Antigenic preparations from Leishmania infantum extracts were isolated by electro-elution from polyacrylamide gels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interferon-gamma is required for the late but not early control of Leishmania amazonensis infection in C57Bl/6 mice

The critical role of interferon-gamma (IFN-gamma) in the resistance of C57Bl/6 mice to Leishmania major is widely established but its role in the relative resistance of these animals to L. amazonensis infection is still not clear. In this work we use C57Bl/6 mice congenitally deficient in the IFN-gamma gene (IFN-gamma KO) to address this issue. We found that IFN-gamma KO mice were as resistant as their wild-type (WT) counterparts at least during the first two months of infection. Afterwards, whereas WT mice maintained lesion growth under control, IFN-gamma KO mice developed devastating lesions. At day 97 of infection, their lesions were 9-fold larger than WT controls, concomitant with an increased parasite burden. At this stage, lesion-draining cells from IFN-gamma KO mice had impaired capacity to produce interleukin-12 (IL-12) and tumour necrosis factor-a in response to parasite antigens whereas IL-4 was slightly increased in comparison to infected WT mice. Together, these results show that IFN-gamma is not critical for the initial control of L. amazonensis infection in C57Bl/6 mice, but is essential for the development of a protective Th1 type immune response in the later stages.     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Induction of autophagy correlates with increased parasite load of Leishmania amazonensis in BALB/c but not C57BL/6 macrophages

We investigated the role of autophagy in infection of macrophages by Leishmania amazonensis. Induction of autophagy by IFN-γ or starvation increased intracellular parasite load and the percentages of infected macrophages from BALB/c but not from C57BL/6 mice. In contrast, starvation did not affect the replication of either Leishmania major or Trypanosoma cruzi in BALB/c macrophages. In BALB/c macrophages, starvation resulted in increased monodansylcadaverine staining and in the appearance of double-membrane and myelin-like vesicles characteristic of autophagosomes. Increased parasite load was associated with a reduction in NO levels and was attenuated by wortmannin, an inhibitor of autophagy. In infected macrophages from BALB/c, but not from C57BL/6 mice, starvation increased the number of lipid bodies and the amounts of PGE₂ produced. Exogenous PGE₂ increased parasite load in macrophages from BALB/c, but not C57BL/6 mice. The cyclooxygenase inhibitor indomethacin prevented the increase of parasite load in starved BALB/c macrophages, and actually induced parasite killing. These results suggest that autophagy regulates the outcome of L. amazonensis infection in macrophages in a host strain specific manner.     

Differences in anxiety-related behavior between apolipoprotein E-deficient C57BL/6 and wild type C57BL/6 mice

The influence of ApoE gene deletion on the anxiety state has not been previously investigated. The elevated plus maze was used in this study to determine differences in anxiety-related behavior between apoE-deficient and wild type C57BL/6 mice. The apoE-deficient mice demonstrated less anxiety on the elevated plus maze by spending more time in the open arms of the elevated plus maze compared to wild type mice (p<0.001). Additionally, female apoE-deficient mice visited the open arm of the maze more often than their apoE-deficient male counterpart (p<0.05). The anxiety state and/or sex are possible variables to be considered when designing physiological and/or behavioral studies involving mice that are apoE-deficient.     

Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis

The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni) against Leishmania (Leishmania) amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography–mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L.) amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg^-1.day^-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L.) amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-β and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment.     

Coxiella burnetii infection in C57BL/6 mice aged 1 or 14 months

The objective of this study was to investigate the effects of age on infection with Coxiella burnetii, the agent of Q fever. Bacterial burden and granuloma number were increased in the spleens of 14-month-old as compared with 1-month-old mice. This increase was not the result of an anti-inflammatory macrophage response, because inflammatory and anti-inflammatory cytokines were induced in macrophages from young mice but were repressed in mature mice. In addition, macrophage microbicidal competence was similar in mature and young mice. These results suggest the importance of individual host factors in the pathophysiology of an infectious disease such as Q fever.     

Leishmania amazonensis infection in nude mice.

Leishmania amazonensis is an intracellular protozoan parasite of macrophages. Cutaneous leishmaniasis in an immunocompetent host begins as papules or nodules followed by ulceration at the site of promastigote inoculation. In this study, the pathological changes of cutaneous leishmaniasis lesions in T cell deficient nude mice were examined. When infected with L. amazonensis promastigotes, nude mice developed non-ulcerative cutaneous nodules. By histological examination of cutaneous lesions, massive accumulation of vacuolated histiocytes containing amastigotes was observed in all the nude mice. Although infiltration of mononuclear and polymorphonuclear cells was seen in the lesions of immunocompetent mice, few such cells were observed in the lesions of nude mice. These results indicate the importance of T cells on the ulcer formation in cutaneous leishmaniasis.     

Delayed matching-to-position performance in C57BL/6N mice

Delayed matching-to-sample is one of the most frequently employed behavioral tasks for assessing spatial working memory in animals. Although the advantages of the task have been widely acknowledged and it is used in the study of a variety of species, its application to mice has been rare. In the present study, we reported the efficacy of a delayed matching-to-position task in C57BL mice lever-pressing in an operant-conditioning chamber. Each trial started with the press of a back lever, followed by the presentation of either a left or right front lever. When the ratio requirement for presses to the front lever (sample) was met, a delay interval started. Delay interval continued until the mice made the first response after the elapse of the programmed delay interval. This was followed by the presentation of a choice of left or right front levers. The choice of the same front lever as the sample was reinforced, whereas the other was not. The proportion of correct choices showed a delay-dependent decrement. A higher ratio of response requirement to the sample resulted in increased accuracy, but the duration of the intertrial interval had no effect. Preceding trials also influenced response accuracy, indicating proactive interference. Overall, the results replicated the effects of parametric manipulations reported in other species, and thus, our findings validate the efficacy of the task for assessing spatial working memory in laboratory mice.     

Substrains matter in phenotyping of C57BL/6 mice

The inbred mouse strain C57BL/6 has been widely used as a background strain for spontaneous and induced mutations. Developed in the 1930s, the C57BL/6 strain diverged into two major groups in the 1950s, namely, C57BL/6J and C57BL/6N, and more than 20 substrains have been established from them worldwide. We previously reported genetic differences among C57BL/6 substrains in 2009 and 2015. Since then, dozens of reports have been published on phenotypic differences in behavioral, neurological, cardiovascular, and metabolic traits. Substrains need to be chosen according to the purpose of the study because phenotypic differences might affect the experimental results. In this paper, we review recent reports of phenotypic and genetic differences among C57BL/6 substrains, focus our attention on the proper use of C57BL/6 and other inbred strains in the era of genome editing, and provide the life science research community wider knowledge about this subject.     

Estrogen and progesterone affect responses to malaria infection in female C57BL/6 mice

Background: Previous data from our laboratory suggest that gonadally intact C57BL/6 male mice are more likely than their female counterparts to die from Plasmodium chabaudi infection, to recover more slowly from weight loss and hematocrit loss, and to have reduced interferon-γ (IFN-γ) and interleukin-10 (IL-10) responses. Removal of the ovaries, and hence, the primary production of sex steroids in females, reverses these differences.Objective: We hypothesized that sex differences in response to P chabaudi may be mediated by differential synthesis of IFN-γ and IL-10 that is influenced by estrogen, progesterone, or both.Methods: C57BL/6 female mice (n = 200; n = 10/time point/treatment/experiment) were ovariectomized and implanted with a 21-day controlled-release pellet containing either 0.1 mg of 17β-estradiol (E₂), 10 mg of progesterone (P₄), 0.1 mg of E₂ plus 10 mg of P₄, or cholesterol (placebo). Females were inoculated with 10⁶P chabaudi-infected erythrocytes. Body mass, body temperature, hematocrit, parasitemia, cytokine production, and antibody responses were monitored 0, 3, 5, 7, 10, 14, and 21 days postinoculation.Results: Administration of E₂, either alone or in combination with P₄, mitigated infection-induced weight loss, hematocrit loss, and hypothermia, compared with females receiving placebo pellets (P < 0.05 in each case). Hormone treatment did not affect levels of parasitemia. Females administered E₂ alone or in combination with P₄ produced 4 to 7 times higher IFN-γ and IL-10 during peak parasitemia than did females implanted with pellets containing either P₄ alone or placebo (P < 0.05 in each case). Exposure to E₂, either alone or in combination with P₄, increased anti-P chabaudi immunoglobulin G (IgG1) responses and the ratio of IgG1 to IgG2c (P < 0.05 in each case).Conclusion: This animal study suggests that physiological levels of estrogen, rather than progesterone, enhance immunity and, possibly, protect females from disease symptoms during malaria infection.     

Mast cells and macrophages in normal C57/BL/6 mice

Mast cells and macrophages have an important role in immunity and inflammation. Because mice are used extensively for experimental studies investigating immunological and inflammatory responses, we examined mast cell and macrophage distribution in normal murine tissues. Mast cells were abundant in the murine dermis, tongue, and skeletal muscle but were rarely found in the heart, lung, spleen, kidney, liver, and the bowel mucosa. In contrast, dogs exhibited large numbers of mast cells in the lung parenchyma, liver, and bowel. Some murine dermal mast cells had long cytoplasmic projections filled with granular content. Mouse mast cells demonstrated intense histamine immunoreactivity and were identified with histochemical enzymatic techniques for tryptase and chymase. Macrophages, identified using the monoclonal antibody F4/80, were abundant in the spleen, lung, liver, kidney, and bowel but relatively rare in the heart, tongue, and dermis. Using a nuclease protection assay we investigated mRNA expression of stem cell factor (SCF), a crucial survival factor for mast cells, and the macrophage growth factors macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Stem cell factor mRNA was highly expressed in the murine lung. Relatively low levels of SCF mRNA expression were found in the tongue and earlobe, which are tissues containing a high number of mast cells. Macrophage CSF and GM-CSF mRNA was highly expressed in the lung and spleen. The murine heart, an organ with a low macrophage content, expressed high levels of M-CSF but negligible levels of GM-CSF mRNA. Constitutive growth factor mRNA expression in murine tissues without significant populations of mast cells and macrophages may suggest an alternative role for these factors in tissue homeostasis.     

Mechanisms of depression of splenic natural killer cell function in C57BL/6 mice infected with Leishmania donovani

C57BL/6 mice chronically infected with the protozoan parasite Leishmania donovani exhibit profoundly depressed splenic natural killer (NK) cell activity as measured by in vitro cytolysis of lymphoma target cells. Injection of infected mice with an interferon (IFN) inducer or in vitro treatment of infected splenocytes with IFN, a phorbol ester, or indomethacin failed to restore their NK activity to the degree shown by age-matched, uninfected mice. Fractionation of infected splenocytes by nylon wool, Sephadex G-10, or carbonyl iron and magnetism treatments was also unable to effect an increase in NK activity. Addition of infected splenocytes to uninfected ones in in vitro NK assays suppressed the NK activity of the latter, and the suppression could be partially or wholly abrogated by prior fractionation of infected splenocytes by the methods noted above. In vitro treatment of infected splenocytes with concanavalin A revealed the presence of NK activity in these cell populations. The results indicate that splenocytes in L. donovani-infected mice become insensitive to IFN stimulation; and the impairment of another, possibly IFN-independent pathway of NK-cell activation may also contribute to the observed L. donovani-induced depression in splenic NK activity in C57BL/6 mice.