Experimental Evidence for Evolved Tolerance to Avian Malaria in a Wild Population of Low Elevation Hawai‘i ‘Amakihi (Hemignathus virens)

Introduced vector-borne diseases, particularly avian malaria (Plasmodium relictum) and avian pox virus (Avipoxvirus spp.), continue to play significant roles in the decline and extinction of native forest birds in the Hawaiian Islands. Hawaiian honeycreepers are particularly susceptible to avian malaria and have survived into this century largely because of persistence of high elevation refugia on Kaua‘i, Maui, and Hawai‘i Islands, where transmission is limited by cool temperatures. The long term stability of these refugia is increasingly threatened by warming trends associated with global climate change. Since cost effective and practical methods of vector control in many of these remote, rugged areas are lacking, adaptation through processes of natural selection may be the best long-term hope for recovery of many of these species. We document emergence of tolerance rather than resistance to avian malaria in a recent, rapidly expanding low elevation population of Hawai‘i ‘Amakihi (Hemignathus virens) on the island of Hawai‘i. Experimentally infected low elevation birds had lower mortality, lower reticulocyte counts during recovery from acute infection, lower weight loss, and no declines in food consumption relative to experimentally infected high elevation Hawai‘i ‘Amakihi in spite of similar intensities of infection. Emergence of this population provides an exceptional opportunity for determining physiological mechanisms and genetic markers associated with malaria tolerance that can be used to evaluate whether other, more threatened species have the capacity to adapt to this disease.Keywords: Tolerance, Climate change, Hawai‘i ‘Amakihi, Adaptation, Plasmodium relictum, Avian malaria, Honeycreepe

The Anti-Inflammatory Drug Leflunomide Is an Agonist of the Aryl Hydrocarbon Receptor

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity and biological activity of dioxins and related chemicals. The AhR influences a variety of processes involved in cellular growth and differentiation, and recent studies have suggested that the AhR is a potential target for immune-mediated diseases.During a screen for molecules that activate the AhR, leflunomide, an immunomodulatory drug presently used in the clinic for the treatment of rheumatoid arthritis, was identified as an AhR agonist. We aimed to determine whether any biological activity of leflunomide could be attributed to a previously unappreciated interaction with the AhR. The currently established mechanism of action of leflunomide involves its metabolism to A771726, possibly by cytochrome P450 enzymes, followed by inhibition of de novo pyrimidine biosynthesis by A771726. Our results demonstrate that leflunomide, but not its metabolite A771726, caused nuclear translocation of AhR into the nucleus and increased expression of AhR-responsive reporter genes and endogenous AhR target genes in an AhR-dependent manner. In silico Molecular Docking studies employing AhR ligand binding domain revealed favorable binding energy for leflunomide, but not for A771726. Further, leflunomide, but not A771726, inhibited in vivo epimorphic regeneration in a zebrafish model of tissue regeneration in an AhR-dependent manner. However, suppression of lymphocyte proliferation by leflunomide or A771726 was not dependent on AhR.These data reveal that leflunomide, an anti-inflammatory drug, is an agonist of the AhR. Our findings link AhR activation by leflunomide to inhibition of fin regeneration in zebrafish. Identification of alternative AhR agonists is a critical step in evaluating the AhR as a therapeutic target for the treatment of immune disorders

Virtual Embryo: Cell-Agent Based Modeling of Developmental Processes and Toxicities

Presented at BoS

Elucidating an Adverse Outcome Pathway of Microcephaly for Use in Computational Toxicology

Presented at the Annual Society of Toxicology meetin

Activation of AhR target genes by leflunomide requires Arnt.

<p>(A) Induction of the AhR target genes CYP1A1, UGT1A1, and NQO1 was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013128#pone-0013128-g002" target="_blank">figure 2</a>. Vehicle (V, 0.1% DMSO), TCDD (T, 1 nM), leflunomide (L20, 20 µM; L10, 10 µM). GAPDH expression was used as a control. PCR cycle numbers are indicated. Target gene induction in Hepa1 vT{2} cells expressing a functional Arnt protein was similar as to that seen with WT Hepa1 cells. CYP1A1 and NQO1 were not induced by leflunomide in Hepa1 C4 cells that do not express a functional Arnt protein. (B–C) XRE-Luc reporter gene assays in Hepa1 vT{2} and C4 cells. Cells were transfected and treated with leflunomide or controls (VEH, Vehicle, 0.1% v/v DMSO; TCDD, 1 nM; and LEF, leflunomide,10 µM) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013128#s2" target="_blank">methods</a> section. (B) Consistent with semi quantitative RT-PCR analysis, TCDD and leflunomide induced expression of the XRE-Luc reporter gene in Hepa1 vT{2} cells. (C) Treatment with TCDD or leflunomide failed to activate the XRE-Luc reporter in the Hepa1 C4 cells. However, transient co-expression of Arnt rescued XRE-Luc reporter gene induction. Reporter gene assays are the mean ± SEM of three independent experiments.</p

Primer sequences for RT-PCR experiments.

<p><b>H: Human M: Mouse, FP: Forward Primer, RP: Reverse Primer.</b></p><p><b>¹</b><b>Sequences described previously.</b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013128#pone.0013128-Bisson1" target="_blank">[<b>33</b>]</a></p

Leflunomide, but not its active metabolite, A771726, activates the AhR.

<p>(A) Structures of leflunomide (left) and its metabolite A771726 (right). (B–C) Reporter gene assays were conducted in Hepa1.1 cells or in HepG2 cells transiently transfected with XRE-Luc reporter gene. Results are the mean ± SEM of at least three independent experiments, each of which consisted of at least three biological replicates. ***: p<0.001 compared with vehicle treatment and p<0.05 compared with corresponding dose of A771726. (D) To confirm the observations of the reporter gene assays, we performed semi quantitative RT-PCR in WT Hepa1 cells for CYP1A1 with total RNA isolated from cells treated with vehicle (0.1% DMSO), leflunomide (L, 20 µM) or A771726 (M, 20 µM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013128#pone-0013128-g002" target="_blank">Figure 2</a>. GAPDH was included as a control. Consistent with reporter gene assays, A771726 failed to activate CYP1A1 beyond that of vehicle treatment, while leflunomide induced strong CYP1A1 expression. (E) Cellular localization of AhR was analyzed by immunofluorescence of Hepa1 cells treated with TCDD, leflunomide, or A771726 for 1 or 3 hours. The FITC (green) channel represents AhR staining, while DAPI (blue) represents the nucleus. The AhR translocated to the nucleus following treatment with both TCDD and leflunomide, while it remained in the cytosol following treatment with A771726. (F) M2 is the major tautomeric form of A771726. Molecular docking of M2 and leflunomide in the homology models of mouse (G) and human (H) AhR ligand binding domain reveal favorable energetic and docking for leflunomide but not M2.</p