Douglas E. Jones

Early enhanced Th1 response after Leishmania amazonensis infection of C57BL/6 interleukin-10-deficient mice does not lead to resolution of infection.

ABSTRACT C3H and C57BL/6 mice are resistant to Leishmania major but develop chronic lesions with persistent parasite loads when they are infected with Leishmania amazonensis. These lesions develop in the absence of interleukin-4 (IL-4), indicating that susceptibility to this parasite is not a result of development of a Th2 response. Expression of the cytokine IL-10 during infection could account for the lack of IL-12 expression and poor cell-mediated immunity towards the parasite. Therefore, we tested the hypothesis that IL-10 plays a central role in downmodulating the Th1 response after L. amazonensis infection. Infection of C57BL/6 IL-10-deficient mice indicated that in the absence of IL-10 there was early enhancement of a Th1 response, which was downregulated during the more chronic stage of infection. In addition, although there were 1- to 2-log reductions in the parasite loads within the lesions, the parasites continued to persist, and they were associated with chronic lesions whose size was similar to that of the control lesions. These experiments indicated that L. amazonensis resistance to killing in vivo is only partially dependent on expression of host IL-10. However, IL-10-deficient mice had an enhanced delayed-type hypersensitivity response during the chronic phase of infection, indicating that there were Th1 type effector cells in vivo at this late stage of infection. These results indicate that although IL-10 plays a role in limiting the Th1 response during the acute infection phase, other immunomodulatory factors are responsible for limiting the Th1 response during the chronic phase.

Programmed Death 1–Mediated T Cell Exhaustion during Visceral Leishmaniasis Impairs Phagocyte Function

Control of Leishmania infantum infection is dependent upon Th1 CD4+ T cells to promote macrophage intracellular clearance of parasites. Deficient CD4+ T cell effector responses during clinical visceral leishmaniasis (VL) are associated with elevated production of IL-10. In the primary domestic reservoir of VL, dogs, we define occurrence of both CD4+ and CD8+ T cell exhaustion as a significant stepwise loss of Ag-specific proliferation and IFN-γ production, corresponding to increasing VL symptoms. Exhaustion was associated with a 4-fold increase in the population of T cells with surface expression of programmed death 1 (PD-1) between control and symptomatic populations. Importantly, exhausted populations of CD8+ T cells and to a lesser extent CD4+ T cells were present prior to onset of clinical VL. VL-exhausted T cells did not undergo significant apoptosis ex vivo after Ag stimulation. Ab block of PD-1 ligand, B7.H1, promoted return of CD4+ and CD8+ T cell function and dramatically increased reactive oxygen species production in cocultured monocyte-derived phagocytes. As a result, these phagocytes had decreased parasite load. To our knowledge, we demonstrate for the first time that pan-T cell, PD-1–mediated, exhaustion during VL influenced macrophage-reactive oxygen intermediate production. Blockade of the PD-1 pathway improved the ability of phagocytes isolated from dogs presenting with clinical VL to clear intracellular parasites. T cell exhaustion during symptomatic canine leishmaniasis has implications for the response to vaccination and therapeutic strategies for control of Leishmania infantum in this important reservoir species.

Immunologic Indicators of Clinical Progression during Canine Leishmania infantum Infection

ABSTRACT In both dogs and humans Leishmania infantum infection is more prevalent than disease, as infection often does not equate with clinical disease. Previous studies additively indicate that advanced clinical visceral leishmaniasis is characterized by increased production of anti-Leishmania antibodies, Leishmania-specific lymphoproliferative unresponsiveness, and decreased production of gamma interferon (IFN-γ) with a concomitant increase of interleukin-10 (IL-10). In order to differentiate infection versus progressive disease for better disease prognostication, we temporally evaluated humoral and cellular immunologic parameters of naturally infected dogs. The work presented here describes for the first time the temporal immune response to natural autochthonous L. infantum infection in foxhounds within the United States. Several key changes in immunological parameters should be considered when differentiating infection versus clinical disease, including a dramatic rise in IgG production, progressive increases in antigen-specific peripheral blood mononuclear cell proliferation, and IFN-γ production. Polysymptomatic disease is precluded by increased IL-10 production and consistent detection of parasite kinetoplast DNA in whole blood. This clinical presentation and the immuno-dysregulation mirror those observed in human patients, indicating that this animal model will be very useful for testing immunomodulatory anti-IL-10 and other therapies.

The Immunology of Leishmania Infection and the Implications for Vaccine Development

Abstract: Leishmania parasites are vector‐borne protozoal pathogens found in tropical and subtropical regions of both the Old and New World. These parasites can cause visceral or cutaneous disease, and the pathology of the infection is determined by both host immune factors and species/strain differences of the parasite. Dogs are an important reservoir for maintaining the population of Leishmania parasites that can lead to visceral leishmaniasis in humans, and a vaccination approach may be an effective method for reducing the numbers of infected dogs. Resistance to leishmaniasis has been consistently associated with a T helper 1 immune response, characterized by the production of IFN‐gamma by the antigen‐specific lymphocyte population. The development of this Th1 response has been shown to be dependent upon both cytokines and dendritic cells during T cell activation. However, the development of a Leishmania vaccine effective in preventing these chronic diseases has proven to be a challenge. Vaccine trials have focused on whole‐killed or subunit vaccines with adjuvants. Newer experimental strategies involve the attenuation of the Leishmania parasite via gene deletion technologies or the expression of specific Leishmania peptides within attenuated organisms, such as Bacillus Calmette Guérin. DNA vaccines and dendritic cell potentiators, such as CpG oligodeoxynucleotides and Flt‐3 ligand, are also in the early stage of development. In addition, as part of blocking the transmission cycle of leishmaniasis, several laboratories are also exploring the possibility of immunomodulating the host toward the bite of the sand fly.

Altered dendritic cell phenotype in response to Leishmania amazonensis amastigote infection is mediated by MAP kinase, ERK.

Initiation of productive immune responses against Leishmania depends on the successful transition of dendritic cells (DC) from an immature to a mature phenotype. This process is characterized by high CD40 surface expression as well as interleukin-12 production, which are frequently seen in response to L. major infection. In vivo footpad infection of C3HeB/FeJ mice for 7 days with L. amazonensis promoted an immature CD11c(+) DC phenotype characterized by both significantly low CD40 surface expression and significantly decreased interleukin-12p40 production compared with L. major infection of these same mice. In vitro infection of bone marrow-derived dendritic cells with L. amazonensis amastigotes resulted in rapid and significant phosphorylation of the mitogen activated protein kinase, extracellular signal-regulated kinase 1/2, observed within minutes of exposure to the parasite. Infection with L. amazonensis promastigotes led to increased 1/2 phosphorylation after 4 hours of infection compared with L. major infection, which correlated with promastigote transformation into amastigotes. Treatment of bone marrow-derived dendritic cells with a mitogen activated protein kinase kinase-specific inhibitor, PD98059, led to regained surface CD40 expression and interleukin-12p40 production following L. amazonensis amastigote infection compared with non-treated, infected DC. Treatment of L. amazonensis-infected mice with the highly-specific mitogen activated protein kinase kinase inhibitor, CI-1040, enhanced surface CD40 expression on CD11c(+) DC obtained from the draining lymph node. L. amazonensis amastigotes, through activation of extracellular signal-regulated kinase 1/2, inhibit the ability of DC to undergo proper maturation both in vitro and in vivo.

Mathematical modelling of immune response in tissues

We have developed a spatial–temporal mathematical model (PDE) to capture fundamental aspects of the immune response to antigen. We have considered terms that broadly describe intercellular communication, cell movement, and effector function (activation or inhibition). The PDE model is robust to variation in antigen load and it can account for (1) antigen recognition, (2) an innate immune response, (3) an adaptive immune response, (4) the elimination of antigen and subsequent resolution of the immune response or (5) equilibrium of the immune response to the presence of persistent antigen (chronic infection) and the formation of a granuloma.

Parameter estimation and sensitivity analysis in an agent-based model of Leishmania major infection

Computer models of disease take a systems biology approach toward understanding host-pathogen interactions. In particular, data driven computer model calibration is the basis for inference of immunological and pathogen parameters, assessment of model validity, and comparison between alternative models of immune or pathogen behavior. In this paper we describe the calibration and analysis of an agent-based model of Leishmania major infection. A model of macrophage loss following uptake of necrotic tissue is proposed to explain macrophage depletion following peak infection. Using Gaussian processes to approximate the computer code, we perform a sensitivity analysis to identify important parameters and to characterize their influence on the simulated infection. The analysis indicates that increasing growth rate can favor or suppress pathogen loads, depending on the infection stage and the pathogens ability to avoid detection. Subsequent calibration of the model against previously published biological observations suggests that L. major has a relatively slow growth rate and can replicate for an extended period of time before damaging the host cell.

CD4+ Th1 Cells Induced by Dendritic Cell-Based Immunotherapy in Mice Chronically Infected with Leishmania amazonensis Do Not Promote Healing

ABSTRACT The susceptibility of mice to Leishmania amazonensis infection is thought to result from an inability to develop a Th1 response. Our data show that the low levels of gamma interferon (IFN-γ) produced by the draining lymph node (DLN) cells of chronically infected mice could be enhanced in vitro and in vivo with L. amazonensis antigen-pulsed bone marrow-derived dendritic cells (BM-DC) and the Th1-promoting cytokine interleukin-12 (IL-12). Given intralesionally to chronically infected mice, this treatment induced the upregulation of mRNA levels for IFN-γ, the transcription factor T-box expressed in T cells, and IL-12 receptor β2 in CD4+ T cells from the DLN and an increase in parasite-specific immunoglobulin G2a in the serum. However, this Th1 response was not associated with healing, and the antigen-specific enhancement of IFN-γ production remained impaired in the DLN. However, addition of IL-12 to the in vitro recall response was able to recover this defect, suggesting that antigen-presenting cell-derived IL-12 production may be limited in infected mice. This was supported by the fact that L. amazonensis amastigotes limited the production of IL-12p40 from BM-DC in vitro. Altogether, our data indicate that the immune response of mice chronically infected with L. amazonensis can be enhanced towards a Th1 phenotype but that the presence of Th1 CD4+ T cells does not promote healing. This suggests that the phenotype of the CD4+ T cells may not always be indicative of protection to L. amazonensis infection. Furthermore, our data support growing evidence that antigen-presenting cell function, such as IL-12 production, may limit the immune response in L. amazonensis-infected mice.

Antigen-responsive CD4+ T cells from C3H mice chronically infected with Leishmania amazonensis are impaired in the transition to an effector phenotype.

ABSTRACT C3HeB/FeJ mice challenged with Leishmania major develop a polarized Th1 response and subsequently heal, whereas Leishmania amazonensis challenge leads to chronic lesions with high parasite loads at 10 weeks postinfection. In this study, a comparison of draining lymph node cells from L. amazonensis- and L. major-infected mice at 10 weeks postinfection showed equivalent percentages of effector/memory phenotype CD44hi CD4+ T cells producing interleukin-2 (IL-2) and proliferating after antigen stimulation. However, these cells isolated from L. amazonensis-infected mice were not skewed toward either a Th1 or Th2 phenotype in vivo, as evidenced by their unbiased Th1/Th2 transcription factor mRNA profile. In vivo antigen stimulation with added IL-12 failed to enhance gamma interferon (IFN-γ) production of CD4+ T cells from L. amazonensis-infected mice. Antigen stimulation of CD4+ T cells from L. amazonensis-infected mice in vitro in the presence of IL-12 resulted in production of only 10 to 15% of the IFN-γ produced by T cells from L. major-infected mice under identical conditions. These results suggest that the CD4+ T-cell response during chronic L. amazonensis infection is limited during the transition from an early activated CD4+ T-cell population to an effector cell population and demonstrate that these T cells have an intrinsic defect beyond the presence or absence of IL-12 during antigen stimulation.

Activation of peripheral blood monocytes results in more robust production of IL-10 in neonatal foals compared to adult horses.

Foals are particularly vulnerable to infection by Rhodococcus equi during the first 2 weeks of life whereas mature horses are not. While an innate immunodeficiency likely accounts for this clinically relevant vulnerability, the factors that contribute to infection by R. equi have not been fully elucidated. In this study, we demonstrate that cells of the monocyte lineage, including monocytes, macrophages, and dendritic cells, that have been activated with LPS and IFN-gamma, respond with a statistically significant, greater amount of cytokine mRNA production of IL-10, IL-12p35, and IL-12p40 than unstimulated control cells. Interestingly, activation of neonatal cells resulted in a twofold log increase in baseline cytokine mRNA expression of IL-10 compared with adult cells. In contrast, no significant differences in mean cytokine mRNA expression of IL-12p35 and IL-12p40 were detected, suggesting that the defect in chromosomal remodeling that prevents IL-12p35 gene transcription as a cause for decreased IL-12 synthesis in human neonates is not a likely occurrence in equine neonates. Collectively, these differences indicate that in vivo activation of equine cells of the monocyte lineage may result in different autocrine and paracrine cellular responses that vary according to age, with potential impact on regulation of adaptive and innate immune responses.