Omar A. Saldarriaga

Reduced Nitric Oxide Synthase 2 (NOS2) Promoter Activity in the Syrian Hamster Renders the Animal Functionally Deficient in NOS2 Activity and Unable to Control an Intracellular Pathogen

Progressive disease in the hamster model of visceral leishmaniasis, caused by Leishmania donovani, in contrast to infection in mice, mimics the progressive disease observed in untreated humans. During progressive infection in hamsters, there was a vigorous type 1 cellular immune response, which is typically associated with control of infection, suggesting that there was ineffective IFN-γ-mediated macrophage activation. Indeed, at the site of infection, hamsters did not express NO synthase 2 (NOS2), which is the primary mechanism for control of infection in mice. Furthermore, in striking contrast to mouse macrophages, IFN-γ-activated hamster macrophages did not did not express NOS2 nor generate NO, and were unable to restrict the replication of intracellular L. donovani. The absent hamster NOS2 expression was not the result of NOS2 gene deletion and the NOS2 cDNA had an intact open reading frame. Furthermore, the impaired transcription of NOS2 mRNA was selective and not due to global impairment of IFN-γ signaling (members of the IFN-γ-signaling pathway were expressed and functional and IFN-γ up-regulated several primary and secondary response genes). Strikingly, the proximal hamster NOS2 promoter, like the human ortholog, had >20-fold less basal and IFN-γ/LPS-inducible activity than the corresponding mouse promoter. Thus, reduced basal and IFN-γ-induced activity of the hamster NOS2 transcriptional unit, which is unique to this small animal and similar to the human counterpart, accompanies the inability of the animal to control an intracellular pathogen.

Progressive Visceral Leishmaniasis Is Driven by Dominant Parasite-induced STAT6 Activation and STAT6-dependent Host Arginase 1 Expression

The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p<0.001) and increased polyamine synthesis (p<0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p<0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p<0.001) and fibroblasts (p<0.01), and in a hamster fibroblast cell line (p<0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p<0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p<0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection.

Clinical, Parasitologic, and Immunologic Evolution in Dogs Experimentally Infected with Sand Fly-Derived Leishmania chagasi Promastigotes

Experimental infection of dogs with Leishmania infantum has yielded heterogeneous clinical, parasitologic, and immunologic results. We studied dogs infected with 10(5) or 10(4) sand fly-derived promastigotes delivered by the intradermal (ID) or intravenous (IV) routes. Total mortality over 1 year post-infection reached 23.8%. The mortality and proportion of sustained polysymptomatic dogs was highest in the IV-10(5) group. The early appearance of polysymptoms was associated with an increased risk of progression to death. Dissemination of the parasite to lymph nodes was faster, and the subsequent infectivity to sand flies higher, in the IV compared with ID-infected dogs. Parasite-specific IgG1 or IgG2 production was similar among the groups, but higher interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) expression was associated with polysymptomatic dogs. On the basis of the data obtained from this study, a sample size analysis using different endpoints for future vaccine trials is described.

Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis.

BackgroundThe Syrian hamster, Mesocricetus auratus, has distinct immunological features and is uniquely susceptible to intracellular pathogens. Studies in hamsters are limited by the relative unavailability of tools to conduct immunological studies. To address this limitation we developed duplex real-time reverse transcriptase (RT) PCR assays for the relative quantification of the mRNAs of hamster cytokines, chemokines, and related immune response molecules.ResultsReal-time RT-PCR primers and probes were synthesized for analysis of interleukin (IL)-4, IFN-γ, TNF-α, IL-10, IL-12p40, TGF-β, IL-13, IL-21, chemokine ligand (CCL) 22, CCL17, Chemokine (C-C motif) receptor 4 and FoxP3 expression. Standard curves and validation experiments were performed for each real-time RT-PCR assay, allowing us to use the comparative Ct (2-ΔΔCt) method to calculate changes in gene expression. Application of the real-time RT PCR assays to a biological model was demonstrated by comparing mRNA expression in skin and lymph node tissues between uninfected and Leishmania panamensis infected hamsters.ConclusionsThe duplex real-time RT PCR assays provide a powerful approach for the quantification of cytokine transcription in hamsters, and their application to a model of cutaneous leishmaniasis suggests that a balanced type 1 and type 2 cytokine response contributes to the chronic, nonprogressive course of disease. These new molecular tools will further facilitate investigation into the mechanisms of disease in the hamster, not only for models of leishmaniasis, but also for other viral, bacterial, fungal, and parasitic infections.

Growth Factor and Th2 Cytokine Signaling Pathways Converge at STAT6 to Promote Arginase Expression in Progressive Experimental Visceral Leishmaniasis

Host arginase 1 (arg1) expression is a significant contributor to the pathogenesis of progressive visceral leishmaniasis (VL), a neglected tropical disease caused by the intracellular protozoan Leishmania donovani. Previously we found that parasite-induced arg1 expression in macrophages was dependent on STAT6 activation. Arg1 expression was amplified by, but did not require, IL-4, and required de novo synthesis of unknown protein(s). To further explore the mechanisms involved in arg1 regulation in VL, we screened a panel of kinase inhibitors and found that inhibitors of growth factor signaling reduced arg1 expression in splenic macrophages from hamsters with VL. Analysis of growth factors and their signaling pathways revealed that the Fibroblast Growth Factor Receptor 1 (FGFR-1) and Insulin-like Growth Factor 1 Receptor (IGF-1R) and a number of downstream signaling proteins were activated in splenic macrophages isolated from hamsters infected with L. donovani. Recombinant FGF-2 and IGF-1 increased the expression of arg1 in L. donovani infected hamster macrophages, and this induction was augmented by IL-4. Inhibition of FGFR-1 and IGF-1R decreased arg1 expression and restricted L. donovani replication in both in vitro and ex vivo models of infection. Inhibition of the downstream signaling molecules JAK and AKT also reduced the expression of arg1 in infected macrophages. STAT6 was activated in infected macrophages exposed to either FGF-2 or IGF-1, and STAT6 was critical to the FGFR-1- and IGF-1R-mediated expression of arg1. The converse was also true as inhibition of FGFR-1 and IGF-1R reduced the activation of STAT6 in infected macrophages. Collectively, these data indicate that the FGFR/IGF-1R and IL-4 signaling pathways converge at STAT6 to promote pathologic arg1 expression and intracellular parasite survival in VL. Targeted interruption of these pathological processes offers an approach to restrain this relentlessly progressive disease.

Congenital Transmission of Experimental Leishmaniasis in a Hamster Model

Little information is available on transplacental transmission of Leishmania spp. We determined the frequency and impact of congenital infection caused by Leishmania panamensis or L. donovani in experimentally infected hamsters. A polymerase chain reaction showed that congenital transmission occurred in 25.8% (24 of 93) of offspring born to L. panamensis-infected hamsters and 14.6% (11 of 75) offspring born to L. donovani-infected hamsters. Mortality during lactation was higher in offspring born to L. panamensis-infected hamsters and offspring born to L. donovani-infected hamsters than controls, and lymphoproliferation to Leishmania was more frequent in offspring born to L. panamensis-infected hamsters (17.4%, 11 of 63) than in offspring born to L. donovani-infected hamsters (8.5%, 3 of 35). After weaning, only offspring born to L. donovani-infected hamsters had lower weight gain (P < 0.001) and hematocrit levels (P = 0.0045) than controls. Challenge of offspring born to L. panamensis-infected hamsters with L. panamensis showed no differences in lesion evolution, and offspring born to L. donovani-infected hamsters were more susceptible to L. donovani challenge than controls. Consequently, prenatal exposure of hamsters to L. donovani significantly increased the mortality risk and susceptibility to secondary homologous infection.

A novel molecular test to diagnose canine visceral leishmaniasis at the point of care

Dogs are the principal reservoir hosts of zoonotic visceral leishmaniasis (VL) but current serological methods are not sensitive enough to detect all subclinically infected animals, which is crucial to VL control programs. Polymerase chain reaction (PCR) methods have greater sensitivity but require expensive equipment and trained personnel, impairing its implementation in endemic areas. We developed a diagnostic test that uses isothermal recombinase polymerase amplification (RPA) to detect Leishmania infantum. This method was coupled with lateral flow (LF) reading with the naked eye to be adapted as a point-of-care test. The L. infantum RPA-LF had an analytical sensitivity similar to real time-PCR, detecting DNA of 0.1 parasites spiked in dog blood, which was equivalent to 40 parasites/mL. There was no cross amplification with dog or human DNA or with Leishmania braziliensis, Leishmania amazonensis, or Trypanosoma cruzi. The test also amplified Leishmania donovani strains (N = 7). In a group of clinically normal dogs (N = 30), RPA-LF detected more subclinical infections than rK39 strip test, a standard serological method (50% versus 13.3% positivity, respectively; P = 0.005). Also, RPA-LF detected L. infantum in noninvasive mucosal samples of dogs with a sensitivity comparable to blood samples. This novel molecular test may have a positive impact in leishmaniasis control programs.

Attenuation of Intestinal Inflammation in Interleukin-10-Deficient Mice Infected with Citrobacter rodentium

ABSTRACT Interleukin-10 (IL-10) curtails immune responses to microbial infection and autoantigens and contributes to intestinal immune homeostasis, yet administration of IL-10 has not been effective at attenuating chronic intestinal inflammatory conditions, suggesting that its immune functions may be context dependent. To gain a broader understanding of the importance of IL-10 in controlling mucosal immune responses to infectious challenges, we employed the murine attaching and effacing pathogen Citrobacter rodentium, which colonizes primarily the surfaces of the cecum and colon and causes transient mucosal inflammation driven by Th17 and Th1 T helper cells. Infection induced macrophage and dendritic cell production of IL-10, which diminished antibacterial host defenses, because IL-10-deficient mice cleared infection faster than wild-type controls. In parallel, the mice had less acute infection-associated colitis and resolved it more rapidly than controls. Importantly, transient C. rodentium infection protected IL-10-deficient mice against the later development of spontaneous colitis that normally occurs with aging in these mice. Genome-wide expression studies revealed that IL-10 deficiency was associated with downregulation of proinflammatory pathways but increased expression of the anti-inflammatory cytokine IL-27 in response to infection. IL-27 was found to suppress in vitro Th17 and, to a lesser degree, Th1 differentiation independent of IL-10. Furthermore, neutralization of IL-27 resulted in more severe colitis in infected IL-10-deficient mice. Together, these findings indicate that IL-10 is dispensable for resolving C. rodentium-associated colitis and further suggest that IL-27 may be a critical factor for controlling intestinal inflammation and Th17 and Th1 development by IL-10-independent mechanisms.

Identification of hamster inducible nitric oxide synthase (iNOS) promoter sequences that influence basal and inducible iNOS expression.

IFN‐γ/LPS‐activated hamster (Mesocricetus auratus) macrophages express significantly less iNOS (NOS2) than activated mouse macrophages, which contributes to the hamsterˈs susceptibility to intracellular pathogens. We determined a mechanism responsible for differences in iNOS promoter activity in hamsters and mice. The HtPP (1.2 kb) showed low basal and inducible promoter activity when compared with the mouse, and sequences within a 100‐bp region (−233 to −133) of the mouse and hamster promoters influenced this activity. Moreover, within this 100 bp, we identified a smaller region (44 bp) in the mouse promoter, which recovered basal promoter activity when swapped into the hamster promoter. The mouse homolog (100‐bp region) contained a cis‐element for NF‐IL‐6 (−153/−142), which was absent in the hamster counterpart. EMSA and supershift assays revealed that the hamster sequence did not support the binding of NF‐IL‐6. Introduction of a functional NF‐IL‐6 binding sequence into the hamster promoter or its alteration in the mouse promoter revealed the critical importance of this transcription factor for full iNOS promoter activity. Furthermore, the binding of NF‐IL‐6 to the iNOS promoter (−153/−142) in vivo was increased in mouse cells but was reduced in hamster cells after IFN‐γ/LPS stimulation. Differences in the activity of the iNOS promoters were evident in mouse and hamster cells, so they were not merely a result of species‐specific differences in transcription factors. Thus, we have identified unique DNA sequences and a critical transcription factor, NF‐IL‐6, which contribute to the overall basal and inducible expression of hamster iNOS.

An Innovative Field-Applicable Molecular Test to Diagnose Cutaneous Leishmania Viannia spp. Infections

Cutaneous and mucosal leishmaniasis is widely distributed in Central and South America. Leishmania of the Viannia subgenus are the most frequent species infecting humans. L. (V.) braziliensis, L. (V.) panamensis are also responsible for metastatic mucosal leishmaniasis. Conventional or real time PCR is a more sensitive diagnostic test than microscopy, but the cost and requirement for infrastructure and trained personnel makes it impractical in most endemic regions. Primary health systems need a sensitive and specific point of care (POC) diagnostic tool. We developed a novel POC molecular diagnostic test for cutaneous leishmaniasis caused by Leishmania (Viannia) spp. Parasite DNA was amplified using isothermal Recombinase Polymerase Amplification (RPA) with primers and probes that targeted the kinetoplast DNA. The amplification product was detected by naked eye with a lateral flow (LF) immunochromatographic strip. The RPA-LF had an analytical sensitivity equivalent to 0.1 parasites per reaction. The test amplified the principal L. Viannia species from multiple countries: L. (V.) braziliensis (n = 33), L. (V.) guyanensis (n = 17), L. (V.) panamensis (n = 9). The less common L. (V.) lainsoni, L. (V.) shawi, and L. (V.) naiffi were also amplified. No amplification was observed in parasites of the L. (Leishmania) subgenus. In a small number of clinical samples (n = 13) we found 100% agreement between PCR and RPA-LF. The high analytical sensitivity and clinical validation indicate the test could improve the efficiency of diagnosis, especially in chronic lesions with submicroscopic parasite burdens. Field implementation of the RPA-LF test could contribute to management and control of cutaneous and mucosal leishmaniasis.