Advertisement

Differentiation-dependent antiviral capacities of amphibian (Xenopus laevis) macrophages

  • Amulya Yaparla
    Footnotes
    Affiliations
    Department of Biological Sciences, George Washington University, Washington, D. C. 20052-0066
    Search for articles by this author
  • Milan Popovic
    Affiliations
    Department of Biological Sciences, George Washington University, Washington, D. C. 20052-0066
    Search for articles by this author
  • Leon Grayfer
    Correspondence
    Recipient of research support at the new investigator level from the George Washington University Department of Biological Sciences. To whom correspondence should be addressed: Dept. of Biological Sciences, George Washington University, 800 22nd St. NW, Suite 6000, Washington, D. C. 20052-0066. Tel.: 202-994-8076
    Affiliations
    Department of Biological Sciences, George Washington University, Washington, D. C. 20052-0066
    Search for articles by this author
  • Author Footnotes
    1 Recipient of summer research support from the George Washington University Department of Biological Sciences of Teaching and Research Stipends and the Harlan Research Fund.
Open AccessPublished:December 19, 2017DOI:https://doi.org/10.1074/jbc.M117.794065
      Infections by ranaviruses such as Frog virus 3 (Fv3), are significantly contributing to worldwide amphibian population declines. Notably, amphibian macrophages (Mϕs) are important to both the Fv3 infection strategies and the immune defense against this pathogen. However, the mechanisms underlying amphibian Mϕ Fv3 susceptibility and resistance remain unknown. Mϕ differentiation is mediated by signaling through the colony-stimulating factor-1 receptor (CSF-1R) which is now known to be bound not only by CSF-1, but also by the unrelated interleukin-34 (IL-34) cytokine. Pertinently, amphibian (Xenopus laevis) Mϕs differentiated by CSF-1 and IL-34 are highly susceptible and resistant to Fv3, respectively. Accordingly, in the present work, we elucidate the facets of this Mϕ Fv3 susceptibility and resistance. Because cellular resistance to viral replication is marked by expression of antiviral restriction factors, it was intuitive to find that IL-34-Mϕs possess significantly greater mRNA levels of select restriction factor genes than CSF-1-Mϕs. Xenopodinae amphibians have highly expanded repertoires of antiviral interferon (IFN) cytokine gene families, and our results indicated that in comparison with the X. laevis CSF-1-Mϕs, the IL-34-Mϕs express substantially greater transcripts of representative IFN genes, belonging to distinct gene family clades, as well as their cognate receptor genes. Finally, we demonstrate that IL-34-Mϕ–conditioned supernatants confer IFN-mediated anti-Fv3 protection to the virally susceptible X. laevis kidney (A6) cell line. Together, this work underlines the differentiation pathways leading to Fv3-susceptible and -resistant amphibian Mϕ populations and defines the molecular mechanisms responsible for these differences.

      Introduction

      Amphibian population die-offs resulting from the Frog virus 3 (Fv3)
      The abbreviations used are: Fv3
      frog virus 3
      CSF-1
      colony-stimulating factor-1
      IFN
      interferon
      IL
      interleukin
      macrophage
      Bar
      baricitinib
      mo
      morpholino
      MOI
      multiplicity of infection
      qPCR
      quantitative PCR.
      ranavirus (family Iridoviridae) infections are significantly contributing to the worldwide amphibian declines (
      • Carey C.
      • Cohen N.
      • Rollins-Smith L.
      Amphibian declines: an immunological perspective.
      ,
      • Daszak P.
      • Berger L.
      • Cunningham A.A.
      • Hyatt A.D.
      • Green D.E.
      • Speare R.
      Emerging infectious diseases and amphibian population declines.
      ). Although much remains to be learned regarding the facets of amphibian immunity against these viral agents, macrophages (Mϕs) are now known to be integral to both the Fv3 infection strategy and to the amphibian host immune responses against this pathogen (
      • Grayfer L.
      • Andino Fde J.
      • Chen G.
      • Chinchar G.V.
      • Robert J.
      Immune evasion strategies of ranaviruses and innate immune responses to these emerging pathogens.
      ,
      • Grayfer L.
      • Robert J.
      Amphibian macrophage development and antiviral defenses.
      ). Indeed, the capacity of the anuran (frogs and toads) amphibian Xenopus laevis to mount effective anti-Fv3 responses depends on appropriate Mϕ differentiation (
      • Grayfer L.
      • Robert J.
      Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34.
      ,
      • Grayfer L.
      • Robert J.
      Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages.
      ). As Mϕ differentiation depends on the activation of the colony-stimulating factor-1 (CSF-1) receptor, it is compelling that in addition to CSF-1 (
      • Wang T.
      • Hanington P.C.
      • Belosevic M.
      • Secombes C.J.
      Two macrophage colony-stimulating factor genes exist in fish that differ in gene organization and are differentially expressed.
      • Pixley F.J.
      • Stanley E.R.
      CSF-1 regulation of the wandering macrophage: complexity in action.
      ,
      • Hanington P.C.
      • Wang T.
      • Secombes C.J.
      • Belosevic M.
      Growth factors of lower vertebrates: characterization of goldfish (Carassius auratus L.) macrophage colony-stimulating factor-1.
      • Garceau V.
      • Smith J.
      • Paton I.R.
      • Davey M.
      • Fares M.A.
      • Sester D.P.
      • Burt D.W.
      • Hume D.A.
      Pivotal advance: avian colony-stimulating factor 1 (CSF-1), interleukin-34 (IL-34), and CSF-1 receptor genes and gene products.
      ), this receptor may be engaged by the unrelated interleukin-34 (IL-34) (
      • Belosevic M.
      • Hanington P.C.
      • Barreda D.R.
      Development of goldfish macrophages in vitro.
      • Droin N.
      • Solary E.
      Editorial: CSF1R, CSF-1, and IL-34, a “menage a trois” conserved across vertebrates.
      ,
      • Lin H.
      • Lee E.
      • Hestir K.
      • Leo C.
      • Huang M.
      • Bosch E.
      • Halenbeck R.
      • Wu G.
      • Zhou A.
      • Behrens D.
      • Hollenbaugh D.
      • Linnemann T.
      • Qin M.
      • Wong J.
      • Chu K.
      • et al.
      Discovery of a cytokine and its receptor by functional screening of the extracellular proteome.
      • Wei S.
      • Nandi S.
      • Chitu V.
      • Yeung Y.G.
      • Yu W.
      • Huang M.
      • Williams L.T.
      • Lin H.
      • Stanley E.R.
      Functional overlap but differential expression of CSF-1 and IL-34 in their CSF-1 receptor-mediated regulation of myeloid cells.
      ), presumably contributing to the functional heterogeneity seen across vertebrate Mϕs. Our recent findings suggest that whereas the X. laevis Mϕs generated by CSF-1 render animals more susceptible to Fv3, the IL-34-derived Mϕ possess potent antiviral capacities and confer frog resistance to this viral pathogen (
      • Grayfer L.
      • Robert J.
      Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34.
      ,
      • Grayfer L.
      • Robert J.
      Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages.
      ). However, the molecular mechanisms conferring CSF-1-Mϕ susceptibility and IL-34-Mϕ resistance to Fv3 remain to be fully defined.
      Because antiviral interferon (IFN) cytokines represent a major pillar of vertebrate antiviral defenses (
      • Wang T.
      • Hanington P.C.
      • Belosevic M.
      • Secombes C.J.
      Two macrophage colony-stimulating factor genes exist in fish that differ in gene organization and are differentially expressed.
      ,
      • Hanington P.C.
      • Wang T.
      • Secombes C.J.
      • Belosevic M.
      Growth factors of lower vertebrates: characterization of goldfish (Carassius auratus L.) macrophage colony-stimulating factor-1.
      ,
      • Garceau V.
      • Smith J.
      • Paton I.R.
      • Davey M.
      • Fares M.A.
      • Sester D.P.
      • Burt D.W.
      • Hume D.A.
      Pivotal advance: avian colony-stimulating factor 1 (CSF-1), interleukin-34 (IL-34), and CSF-1 receptor genes and gene products.
      • Belosevic M.
      • Hanington P.C.
      • Barreda D.R.
      Development of goldfish macrophages in vitro.
      ), it is particularly notable that amphibians hold a key stage in the evolution of these soluble effectors. Mammals, birds, and reptiles possess three types of IFNs, type I, II, and III IFNs (
      • Sadler A.J.
      • Williams B.R.
      Interferon-inducible antiviral effectors.
      ), of which the type II IFNs mediate a variety of immunological roles, whereas the type I and type III IFNs predominantly participate in antiviral immunity (
      • Zou J.
      • Secombes C.J.
      Teleost fish interferons and their role in immunity.
      ). The type I IFNs of these higher vertebrates are encoded by intronless genes, whereas their type III IFNs (also known as IFNλs/IFNLs) are encoded by five exon/four intron gene transcripts (
      • Zou J.
      • Secombes C.J.
      Teleost fish interferons and their role in immunity.
      ). Conversely, teleost and cartilaginous fish do not appear to possess type III IFNs and encode type I IFNs with five-exon, four-intron organization (
      • Zou J.
      • Secombes C.J.
      Teleost fish interferons and their role in immunity.
      ). Intriguingly, Xenopodinae amphibians possess type I IFN genes with the five-exon, four-intron intron gene organization of the fish counterparts; intronless type I IFNs akin to those seen in higher vertebrates; and intronless and five exon/four intron-containing type III IFN genes (
      • Sang Y.
      • Liu Q.
      • Lee J.
      • Ma W.
      • McVey D.S.
      • Blecha F.
      Expansion of amphibian intronless interferons revises the paradigm for interferon evolution and functional diversity.
      ,
      • Gan Z.
      • Chen S.N.
      • Huang B.
      • Hou J.
      • Nie P.
      Intronless and intron-containing type I IFN genes coexist in amphibian Xenopus tropicalis: insights into the origin and evolution of type I IFNs in vertebrates.
      ). Most notably, we recently demonstrated that X. laevis intron-containing type I and type III IFNs play important roles in anti-Fv3 responses (
      • Grayfer L.
      • De Jesús Andino F.
      • Robert J.
      The amphibian (Xenopus laevis) type I interferon response to frog virus 3: new insight into ranavirus pathogenicity.
      ,
      • Grayfer L.
      • De Jesús Andino F.
      • Robert J.
      Prominent amphibian (Xenopus laevis) tadpole type III interferon response to the frog virus 3 ranavirus.
      • Wendel E.S.
      • Yaparla A.
      • Koubourli D.V.
      • Grayfer L.
      Amphibian (Xenopus laevis) tadpoles and adult frogs mount distinct interferon responses to the Frog Virus 3 ranavirus.
      ).
      Aside from antiviral IFN responses, the capacity of distinct cell types to support or minimize the replication of invading viral pathogens often correlates with their respective gene expression of antiviral restriction factors (
      • Badia R.
      • Angulo G.
      • Riveira-Muñoz E.
      • Pujantell M.
      • Puig T.
      • Ramirez C.
      • Torres-Torronteras J.
      • Marti R.
      • Pauls E.
      • Clotet B.
      • Ballana E.
      • Esté J.A.
      Inhibition of herpes simplex virus type 1 by the CDK6 inhibitor PD-0332991 (palbociclib) through the control of SAMHD1.
      • Ballana E.
      • Esté J.A.
      SAMHD1: at the crossroads of cell proliferation, immune responses, and virus restriction.
      ,
      • Lang F.
      • Li X.
      • Vladimirova O.
      • Hu B.
      • Chen G.
      • Xiao Y.
      • Singh V.
      • Lu D.
      • Li L.
      • Han H.
      • Wickramasinghe J.M.
      • Smith S.T.
      • Zheng C.
      • Li Q.
      • Lieberman P.M.
      • et al.
      CTCF interacts with the lytic HSV-1 genome to promote viral transcription.
      • Li D.J.
      • Verma D.
      • Mosbruger T.
      • Swaminathan S.
      CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription.
      ). Collectively, these cellular proteins present enormous structural and functional diversities and provide the first cellular lines of defense against viral infections by targeting and ablating distinct viral replication mechanisms (
      • Badia R.
      • Angulo G.
      • Riveira-Muñoz E.
      • Pujantell M.
      • Puig T.
      • Ramirez C.
      • Torres-Torronteras J.
      • Marti R.
      • Pauls E.
      • Clotet B.
      • Ballana E.
      • Esté J.A.
      Inhibition of herpes simplex virus type 1 by the CDK6 inhibitor PD-0332991 (palbociclib) through the control of SAMHD1.
      • Ballana E.
      • Esté J.A.
      SAMHD1: at the crossroads of cell proliferation, immune responses, and virus restriction.
      ,
      • Lang F.
      • Li X.
      • Vladimirova O.
      • Hu B.
      • Chen G.
      • Xiao Y.
      • Singh V.
      • Lu D.
      • Li L.
      • Han H.
      • Wickramasinghe J.M.
      • Smith S.T.
      • Zheng C.
      • Li Q.
      • Lieberman P.M.
      • et al.
      CTCF interacts with the lytic HSV-1 genome to promote viral transcription.
      • Li D.J.
      • Verma D.
      • Mosbruger T.
      • Swaminathan S.
      CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription.
      ). Whereas the expression of some of these restriction factors is enhanced by IFNs, others are constitutively expressed (
      • Badia R.
      • Angulo G.
      • Riveira-Muñoz E.
      • Pujantell M.
      • Puig T.
      • Ramirez C.
      • Torres-Torronteras J.
      • Marti R.
      • Pauls E.
      • Clotet B.
      • Ballana E.
      • Esté J.A.
      Inhibition of herpes simplex virus type 1 by the CDK6 inhibitor PD-0332991 (palbociclib) through the control of SAMHD1.
      • Ballana E.
      • Esté J.A.
      SAMHD1: at the crossroads of cell proliferation, immune responses, and virus restriction.
      ,
      • Lang F.
      • Li X.
      • Vladimirova O.
      • Hu B.
      • Chen G.
      • Xiao Y.
      • Singh V.
      • Lu D.
      • Li L.
      • Han H.
      • Wickramasinghe J.M.
      • Smith S.T.
      • Zheng C.
      • Li Q.
      • Lieberman P.M.
      • et al.
      CTCF interacts with the lytic HSV-1 genome to promote viral transcription.
      • Li D.J.
      • Verma D.
      • Mosbruger T.
      • Swaminathan S.
      CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription.
      ).
      To delineate the mechanisms responsible for IL-34–derived Mϕ antiviral resistance, and CSF-1-differentiated Mϕ susceptibility to Fv3, we compared these cell populations for their gene expression of select intron-containing and intronless type I and type III IFNs as well as the respective IFN receptor genes. Additionally, we examined the restriction factor gene expression levels of these Mϕ populations and compared the ability of Fv3 to replicate within CSF- and IL-34–derived Mϕs. Moreover, we examined the ability of CSF-1-Mϕ– and IL-34-Mϕ–conditioned supernatants to restrict Fv3 growth in an otherwise highly Fv3-susceptible X. laevis kidney epithelial cell line (A6).

      Results

      IL-34-Mϕs are more restrictive to Fv3 replication

      We previously demonstrated that X. laevis Mϕs differentiated with IL-34 are resistant to Fv3 infection, whereas CSF-1-derived Mϕs are significantly more susceptible to this virus (
      • Grayfer L.
      • Robert J.
      Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34.
      ,
      • Grayfer L.
      • Robert J.
      Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages.
      ). Accordingly, to examine whether CSF-1-Mϕs support greater Fv3 replication than IL-34-Mϕs, we differentiated IL-34- and CSF-1-Mϕs from X. laevis bone marrow cells, challenged them with Fv3, and assessed the viral loads and expression of Fv3 82R (immediate early, IE), Fv3 95R (delayed early, E), and Fv3 93L (late, L) genes in these respective Mϕ populations. As previously noted, CSF-1-Mϕs possessed significantly greater Fv3 loads than detected in the IL-34-Mϕs (Fig. 1A). Moreover, CSF-1-Mϕs possessed substantially greater magnitudes of Fv3-82R, -95R, and -93L gene expression than detected in IL-34-Mϕs (Fig. 1B). Of the examined Fv3 genes, 93L exhibited the greatest expression differences between CSF-1- and IL-34-Mϕ (Fig. 1B).
      Figure thumbnail gr1
      Figure 1IL-34-Mϕs are more restrictive to Fv3 replication than CSF-1-Mϕs. X. laevis bone marrow cells were cultured in vitro with CSF-1 or IL-34 for 5 days, followed by Fv3 infection at 0.5 MOI for 24 h. A, CSF-1- and IL-34-Mϕ Fv3 DNA loads, determined by absolute qPCR (n = 5). B, FV3-challenged CSF-1- and IL-34-Mϕ gene expression of viral immediate early (82R), delayed early (95R), and late (93L) genes, quantified relative to the gapdh endogenous control (n = 5). Results are means ± S.E. (error bars); *, statistically significant difference (p < 0.05).

      IL-34-Mϕs exhibit greater restriction factor gene expression than CSF-1-Mϕs

      Because our results indicated that IL-34-Mϕs are more restrictive to Fv3 infection while the expression of appropriate restriction factors is crucial to adequate cellular arrest of viral replication (
      • Badia R.
      • Angulo G.
      • Riveira-Muñoz E.
      • Pujantell M.
      • Puig T.
      • Ramirez C.
      • Torres-Torronteras J.
      • Marti R.
      • Pauls E.
      • Clotet B.
      • Ballana E.
      • Esté J.A.
      Inhibition of herpes simplex virus type 1 by the CDK6 inhibitor PD-0332991 (palbociclib) through the control of SAMHD1.
      • Ballana E.
      • Esté J.A.
      SAMHD1: at the crossroads of cell proliferation, immune responses, and virus restriction.
      ,
      • Lang F.
      • Li X.
      • Vladimirova O.
      • Hu B.
      • Chen G.
      • Xiao Y.
      • Singh V.
      • Lu D.
      • Li L.
      • Han H.
      • Wickramasinghe J.M.
      • Smith S.T.
      • Zheng C.
      • Li Q.
      • Lieberman P.M.
      • et al.
      CTCF interacts with the lytic HSV-1 genome to promote viral transcription.
      • Li D.J.
      • Verma D.
      • Mosbruger T.
      • Swaminathan S.
      CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription.
      ), we next examined whether IL-34-Mϕ resistance to Fv3 could be attributed to more robust expression of such restriction factors. In comparison with CSF-1-Mϕs, IL-34-Mϕs exhibited a significantly greater expression levels of the apobec2 (apolipoprotein B mRNA-editing enzyme-2) and trim28 (tripartite motif–containing 28) restriction factors (Fig. 2A), which are known to suppress viral replication (
      • Shi K.
      • Carpenter M.A.
      • Banerjee S.
      • Shaban N.M.
      • Kurahashi K.
      • Salamango D.J.
      • McCann J.L.
      • Starrett G.J.
      • Duffy J.V.
      • Demir Ö.
      • Amaro R.E.
      • Harki D.A.
      • Harris R.S.
      • Aihara H.
      Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B.
      ,
      • Wolf D.
      • Hug K.
      • Goff S.P.
      TRIM28 mediates primer binding site-targeted silencing of Lys1,2 tRNA-utilizing retroviruses in embryonic cells.
      ). Moreover, Fv3 is a dsDNA virus, and our result showed that IL-34-Mϕs also possessed more robust gene expression of samhd1 (SAM domain and HD domain-containing protein 1) and ctcf (CCCTC-binding factor) restriction factors, both of which are known to specifically target dsDNA viruses (
      • Badia R.
      • Angulo G.
      • Riveira-Muñoz E.
      • Pujantell M.
      • Puig T.
      • Ramirez C.
      • Torres-Torronteras J.
      • Marti R.
      • Pauls E.
      • Clotet B.
      • Ballana E.
      • Esté J.A.
      Inhibition of herpes simplex virus type 1 by the CDK6 inhibitor PD-0332991 (palbociclib) through the control of SAMHD1.
      • Ballana E.
      • Esté J.A.
      SAMHD1: at the crossroads of cell proliferation, immune responses, and virus restriction.
      ,
      • Lang F.
      • Li X.
      • Vladimirova O.
      • Hu B.
      • Chen G.
      • Xiao Y.
      • Singh V.
      • Lu D.
      • Li L.
      • Han H.
      • Wickramasinghe J.M.
      • Smith S.T.
      • Zheng C.
      • Li Q.
      • Lieberman P.M.
      • et al.
      CTCF interacts with the lytic HSV-1 genome to promote viral transcription.
      • Li D.J.
      • Verma D.
      • Mosbruger T.
      • Swaminathan S.
      CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription.
      ) (Fig. 2A). By contrast, CSF-1- and IL-34-Mϕs had comparable mRNA levels of cyclophilin B (cypb) and rad21 (Fig. 2A).
      Figure thumbnail gr2
      Figure 2A, bone marrow–derived IL-34-Mϕs exhibit greater restriction factor gene expression than CSF-1-Mϕs. B, peritonea-derived IL-34-Mϕs exhibit greater expression of apobec2 than CSF-1-Mϕs. X. laevis bone marrow and peritonea-derived CSF-1- and IL-34-Mϕ cultures were assessed by qPCR for gene expression of viral restriction factors, with all expression quantified relative to the gapdh endogenous control (n = 5). Results are means ± S.E. (error bars); *, statistically significant difference from CSF-1-Mϕs (p < 0.05).
      We showed previously that, like other vertebrate CSF-1 receptor ligands (
      • Dwyer A.R.
      • Mouchemore K.A.
      • Steer J.H.
      • Sunderland A.J.
      • Sampaio N.G.
      • Greenland E.L.
      • Joyce D.A.
      • Pixley F.J.
      Src family kinase expression and subcellular localization in macrophages: implications for their role in CSF-1-induced macrophage migration.
      ,
      • Grayfer L.
      • Hanington P.C.
      • Belosevic M.
      Macrophage colony-stimulating factor (CSF-1) induces pro-inflammatory gene expression and enhances antimicrobial responses of goldfish (Carassius auratus L.) macrophages.
      ), the frog CSF-1 and IL-34 chemoattract Mϕ precursors and induce their functional differentiation (
      • Grayfer L.
      • Robert J.
      Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34.
      ,
      • Grayfer L.
      • Robert J.
      Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages.
      ). In this respect, intraperitoneal injection of frogs with either recombinant CSF-1 or IL-34 results in peritoneal accumulation of Mϕs that morphologically (85–90%) represent the respective CSF-1- or IL-34-Mϕ populations (
      • Grayfer L.
      • Robert J.
      Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34.
      ,
      • Grayfer L.
      • Robert J.
      Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages.
      ). Moreover, and akin to their bone marrow-derived counterparts, the peritonea-derived IL-34- and CSF-1-Mϕs are respectively highly resistant and susceptible to Fv3 (
      • Grayfer L.
      • Robert J.
      Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34.
      ,
      • Grayfer L.
      • Robert J.
      Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages.
      ). To examine the extent to which restriction factor expression correlates with the Fv3 susceptibility of these Mϕ populations, we examined the expression of the same panel of restriction factor–encoding genes as above, in peritonea-derived CSF-1- and IL-34-Mϕs (Fig. 2B). Of the examined genes and in comparison with peritonea-derived CSF-1-Mϕs, the peritoneal IL-34-Mϕs only expressed greater levels of apobec2 (Fig. 2B).

      IL-34-Mϕs exhibit greater gene expression of intron-containing and intronless type I and type III IFNs than CSF-1-Mϕs

      Xenopodinae encode highly expanded repertoires of phylogenetically distinct intron containing and intronless type I and type III IFNs (ifn, ifnx, ifnl, and ifnlx, respectively (
      • Sang Y.
      • Liu Q.
      • Lee J.
      • Ma W.
      • McVey D.S.
      • Blecha F.
      Expansion of amphibian intronless interferons revises the paradigm for interferon evolution and functional diversity.
      ,
      • Gan Z.
      • Chen S.N.
      • Huang B.
      • Hou J.
      • Nie P.
      Intronless and intron-containing type I IFN genes coexist in amphibian Xenopus tropicalis: insights into the origin and evolution of type I IFNs in vertebrates.
      )). Accordingly, to define the possible mechanisms conferring the disparities in IL-34- and CSF-1-Mϕ-Fv3 resistance, we generated CSF-1- and IL-34-Mϕs, challenged them with Fv3, and examined their gene expression of type I and III IFNs belonging to each of the distinct clades, described in recent reports (
      • Sang Y.
      • Liu Q.
      • Lee J.
      • Ma W.
      • McVey D.S.
      • Blecha F.
      Expansion of amphibian intronless interferons revises the paradigm for interferon evolution and functional diversity.
      ,
      • Gan Z.
      • Chen S.N.
      • Huang B.
      • Hou J.
      • Nie P.
      Intronless and intron-containing type I IFN genes coexist in amphibian Xenopus tropicalis: insights into the origin and evolution of type I IFNs in vertebrates.
      ). In comparison with the CSF-1-Mϕ cultures, IL-34-Mϕs exhibited significantly greater gene expression of intron-containing type I IFNs (ifn1 and ifn7; Fig. 3A), intronless type I IFNs (ifnx2, ifnx6, ifnx11, ifnx13, and ifnx20; Fig. 3, B–D), and intron-containing and intronless type III IFNs (ifnlx1/2, ifnl3, and ifnl4, respectively; Fig. 3, D and E). Virally challenging CSF-1-Mϕ did not result in significant changes to their gene expression of any of the examined IFNs (Fig. 3, A–E). Fv3 challenge completely abrogated the gene expression of IL-34-Mϕ ifn1 (Fig. 3A) and had no significant affect on ifn7, ifnx2, ifnx6, ifnx11, ifnx13, ifnx20, or ifnl4 gene expression (Fig. 3, B–E). By contrast, Fv3-challenged IL-34-Mϕs possessed significantly greater transcript levels of ifnl3 and ifnlx1/2 (Fig. 3, D and E, respectively).
      Figure thumbnail gr3
      Figure 3Gene expression analysis of intron-containing and intronless type I and type III IFNs (IFN, IFNX, IFNL, and IFNLX, respectively) and their receptors in CSF-1- and IL-34-Mϕs. X. laevis bone marrow–derived CSF-1- and IL-34-Mϕs were challenged with Fv3 and examined for gene expression of ifn1 and ifn7 (A); ifnx2 and ifnx6 (B); ifnx11 and ifnx13 (C); ifnx20 and ifnlx1/2 (D); ifnl3 and ifnl4 (E); and ifnar2.1, ifnar2.2, and ifnlr1 (F). All expression was quantified relative to the gapdh endogenous control (n = 5). Results are means ± S.E. (error bars). *, statistical difference from respective CSF-1-Mϕs; * (above horizontal lines), statistical difference between the treatment groups denoted by the respective lines (p < 0.05).

      IL-34-Mϕs express greater levels of type I and type III IFN receptors than CSF-1-Mϕs

      We previously demonstrated that IL-34-Mϕs are more responsive to type I IFN stimulation and inhibit Fv3 replication with greater efficiency than CSF-1-Mϕs (
      • Grayfer L.
      • Andino Fde J.
      • Chen G.
      • Chinchar G.V.
      • Robert J.
      Immune evasion strategies of ranaviruses and innate immune responses to these emerging pathogens.
      ). The type I IFN receptor complex comprises the ligand-binding IFNAR2 chain and the signal-propagating IFNAR1 chain (
      • Droin N.
      • Solary E.
      Editorial: CSF1R, CSF-1, and IL-34, a “menage a trois” conserved across vertebrates.
      ). Conversely, type III IFN receptor complex consists of a ligand-binding IFNLR1 chain and signal-propagating interleukin-10 receptor-2 (IL-10R2) chain (
      • Lin H.
      • Lee E.
      • Hestir K.
      • Leo C.
      • Huang M.
      • Bosch E.
      • Halenbeck R.
      • Wu G.
      • Zhou A.
      • Behrens D.
      • Hollenbaugh D.
      • Linnemann T.
      • Qin M.
      • Wong J.
      • Chu K.
      • et al.
      Discovery of a cytokine and its receptor by functional screening of the extracellular proteome.
      ,
      • Wei S.
      • Nandi S.
      • Chitu V.
      • Yeung Y.G.
      • Yu W.
      • Huang M.
      • Williams L.T.
      • Lin H.
      • Stanley E.R.
      Functional overlap but differential expression of CSF-1 and IL-34 in their CSF-1 receptor-mediated regulation of myeloid cells.
      ). Because IL-34-Mϕ expressed greater levels of all examined IFN genes compared with CSF-1-Mϕ (Fig. 3, A–E), we next examined whether these cells also possessed greater gene expression of type I and III IFN ligand binding chains (ifnar2 and ifnlr1, respectively; Fig. 2F). Notably, X. laevis possess two isoforms of the ifnar2 (ifnar2.1 and ifnar2.2), both of which were significantly more expressed in IL-34-Mϕ than in CSF-1-Mϕ (Fig. 3F). Following Fv3 challenge, IL-34-Mϕs displayed a robust and significant up-regulation of their ifnar2.1, IFNAR2.2, and IFNLR1 gene expression, whereas CSF-1-Mϕs exhibited a modest but significant expression increase in only the ifnar2.2 gene (Fig. 3F).

      IL-34-Mϕ–conditioned supernatants protect frog kidney epithelial cells from Fv3 infection

      The kidneys of anuran amphibians represent a central target for Fv3 replication (
      • Donnelly R.P.
      • Kotenko S.V.
      Interferon-λ: a new addition to an old family.
      ). Because our results indicate that IL-34-Mϕs express greater levels of antiviral IFNs (Fig. 3, A–E), we next examined whether co-incubation of the X. laevis kidney-derived, Fv3-susceptible epithelial cell line (A6) with IL-34-Mϕs or with the supernatants conditioned by these cells (and presumably containing IFNs) would grant A6 cells protection from Fv3. To this end, A6 cells were cultured with CSF-1-Mϕs, IL-34-Mϕs, or medium conditioned by these respective Mϕ populations and infected with Fv3 for 24 h, and Fv3 DNA loads were assessed (Fig. 4A). IL-34-Mϕs (cultured without A6 cells) exhibited substantially reduced Fv3 loads compared with those seen in the CSF-1-Mϕ cultures (Fig. 4A), confirming that IL-34-Mϕs are more resistant to Fv3 than CSF-1-Mϕs. Fv3-infected A6 cells co-cultured with IL-34-Mϕs exhibited modestly (not significantly) reduced Fv3 loads, compared with A6 cultured alone (Fig. 4A). Most notably, A6 cells cultured with IL-34 Mϕ-derived supernatants possessed significantly reduced Fv3 loads compared with those seen in control Fv3-infected A6 cultures (Fig. 4A).
      Figure thumbnail gr4
      Figure 4Supernatants from IL-34 Mϕ cultures confer anti-Fv3 protection to the frog A6 kidney epithelial cell line. A, A6 cells, cultured for 3 h with CSF-1- or IL-34-Mϕs or supernatants (sups) obtained from these respective Mϕ cultures were challenged with Fv3 (0.5 MOI) for 24 h, and Fv3 DNA viral loads were examined by absolute qPCR (n = 5). B, A6 cells were incubated for 3 h with medium conditioned by CSF-1- or IL-34-Mϕ cultures, with or without the Jak1/Tyk2 inhibitor (Bar or PBS, respectively), and infected with Fv3 (0.5 MOI) for 24 h, and Fv3 DNA viral loads were examined by absolute qPCR (n = 5). C, A6 cells were transfected with antisense mo against ifnar2.1/2.2 or ifnlr1, incubated for 3 h with medium conditioned by IL-34-Mϕs, and infected with Fv3 (0.5 MOI) for 24 h before Fv3 DNA viral load analysis (n = 5). D, A6 cells were co-incubated with recombinant extracellular domains of IFNAR2.1 (rAR2) or IFNLR1 (rLR1) and medium conditioned by IL-34-Mϕs for 3 h and infected with Fv3 (0.5 MOI) for 24 h before Fv3 DNA viral load analysis (n = 5). Results are means ± S.E. (error bars). * (above horizontal lines), statistical difference between the treatment groups denoted by the respective lines (p < 0.05).

      IL-34-Mϕ–conditioned supernatants contain IFNs

      The type I and type III IFNs of higher vertebrates signal through distinct receptor complexes but employ the same downstream signaling pathways, including the Janus kinase 1 (Jak1) and tyrosine kinase 2 (Tyk2) (
      • Donnelly R.P.
      • Kotenko S.V.
      Interferon-λ: a new addition to an old family.
      ,
      • Durbin R.K.
      • Kotenko S.V.
      • Durbin J.E.
      Interferon induction and function at the mucosal surface.
      • Kotenko S.V.
      IFN-λs.
      ). Concomitantly, we recently showed that the antiviral effects of both X. laevis type I and type III IFNs could be pharmacologically inhibited with a Jak1/Tyk2 inhibitor, baricitinib (Bar) (
      • Wendel E.S.
      • Yaparla A.
      • Koubourli D.V.
      • Grayfer L.
      Amphibian (Xenopus laevis) tadpoles and adult frogs mount distinct interferon responses to the Frog Virus 3 ranavirus.
      ). To test our hypothesis that the anti-Fv3 A6 cell protection conferred by IL-34-Mϕ–conditioned medium is due to the presence of IFNs within these supernatants, we cultured A6 cells with CSF-1-Mϕ– and IL-34-Mϕ–conditioned media, with or without Bar (Fig. 4B). Bar inhibition of A6 cell IFN signaling significantly abolished the antiviral protection conferred by IL-34-Mϕ supernatants (Fig. 4B), supporting the notion that the anti-Fv3 protection mediated by IL-34-Mϕ–derived supernatants is (at least in part) due to the presence of IFNs within these supernatants.
      To confirm that the anti-Fv3 protection elicited by IL-34-Mϕ supernatants was mediated by antiviral IFNs, we examined the capacity of these supernatants to protect A6 cultures subsequent to their antisense morpholino (mo) knockdown of type I or type III IFN receptor chains (both isoforms of ifnar2.1/2.2 and ifnlr1; Fig. 4C), which are responsible for binding of the respective ligands (
      • Uzé G.
      • Monneron D.
      IL-28 and IL-29: newcomers to the interferon family.
      ,
      • Uzé G.
      • Schreiber G.
      • Piehler J.
      • Pellegrini S.
      The receptor of the type I interferon family.
      ). Pretreating A6 cells with either ifnar2-mo or ifnlr1-mo significantly (but not entirely) reduced the anti-Fv3 protection offered by the IL-34-Mϕs (Fig. 4C).
      As a parallel approach to confirming the antiviral IFN content of IL-34-Mϕ supernatants, we produced the extracellular domains of IFNAR2 (isoform 1) and IFNLR1 in recombinant form (rAR2 and rLR1, respectively) and added these to the A6 cells cultured with IL-34-Mϕ supernatants to bind up any type I or III IFNs, thereby ablating any protection offered by these cytokines (Fig. 4D). The addition of either rAR2 or rLR1 resulted in complete abolishment of the anti-FV3 protection offered by the IL-34-Mϕ supernatants to A6 cultures (Fig. 4D). Culturing A6 cells with either rAR2 or rLR1 alone had no effect on their Fv3 susceptibility (data not shown).

      CSF-1- and IL-34-Mϕs may be counterpolarized

      To discern whether CSF-1 and IL-34 could be functionally counterpolarized with IL-34 and CSF-1, respectively, CSF-1- and IL-34-Mϕs were differentiated for 5 days and counterstimulated for 24 h with IL-34 or CSF-1, respectively, and their IFN ligand and receptor gene expression was examined (Fig. 5, A and B). Akin to our earlier observations (Fig. 3, A–E), IL-34-Mϕ exhibited consistently greater expression of all examined IFNs (ifn7, ifnx13, ifnlx1/2, and ifnl4) as compared with CSF-1-Mϕ (Fig. 5A). Notably, CSF-1-Mϕ stimulated with IL-34 as well as IL-34-Mϕs treated with CSF-1 possessed ifn7, ifnx13, ifnlx1/2, and IFNL4 transcript levels comparable with IL-34-Mϕs (Fig. 5A). Both CSF-1-Mϕs stimulated with IL-34 and IL-34-Mϕs treated with CSF-1 had significantly greater ifnar2.1 (but not ifnar2.2) gene expression levels than those observed in IL-34- or CSF-1-Mϕs (Fig. 5B). By contrast, whereas the CSF-1-Mϕ stimulated with IL-34 had ifnlr1 transcript levels similar to those detected in IL-34-Mϕs, IL-34-Mϕs stimulated with CSF-1 had drastically decreased expression levels of this gene compared with IL-34-Mϕs (Fig. 5B).
      Figure thumbnail gr5
      Figure 5CSF-1-Mϕs may be counterpolarized with IL-34. CSF-1- and IL-34-Mϕs were cultured in vitro for 5 days and stimulated with reciprocal cytokines (IL-34 and CSF-1, respectively) for 24 h. Mϕ cultures were examined for gene expression of ifn7, ifnx13, ifnlx1/2, and ifnl4 (A) and ifnar2.1, ifnar2.2, and ifnlr1 (B). All expression was quantified relative to the gapdh endogenous control (n = 5). Results are means ± S.E. (error bars). *, statistical difference from respective CSF-1-Mϕs; * (above horizontal lines), statistical difference between the treatment groups denoted by the respective lines (p < 0.05).

      Discussion

      Mϕs are integral to the physiology and immune defenses of all vertebrate species. Perhaps thus, many pathogens have coevolved to evade Mϕ immunity and utilize these cells toward their infection strategies. Ranaviruses such as Fv3 represent ecologically and commercially relevant viral pathogens that have espoused this strategy to thrive within their cold-blooded vertebrate hosts (
      • Grayfer L.
      • Andino Fde J.
      • Chen G.
      • Chinchar G.V.
      • Robert J.
      Immune evasion strategies of ranaviruses and innate immune responses to these emerging pathogens.
      ). Fv3 infects, persists, and disseminates within amphibian Mϕs (
      • Grayfer L.
      • Andino Fde J.
      • Chen G.
      • Chinchar G.V.
      • Robert J.
      Immune evasion strategies of ranaviruses and innate immune responses to these emerging pathogens.
      ,
      • Morales H.D.
      • Robert J.
      Characterization of primary and memory CD8 T-cell responses against ranavirus (FV3) in Xenopus laevis.
      ,
      • Robert J.
      • Abramowitz L.
      • Gantress J.
      • Morales H.D.
      Xenopus laevis: a possible vector of Ranavirus infection?.
      ). Whereas Mϕs differentiated by CSF-1 are highly permissive to this Fv3, IL-34-Mϕs not only appear to be resistant to this virus, but may also represent a crucial effector subset in the defense against this pathogen (
      • Grayfer L.
      • Robert J.
      Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34.
      ,
      • Grayfer L.
      • Robert J.
      Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages.
      ). Presently, we show that the means of these cells’ resistance to Fv3 infection and replication reflect their heightened expression of antiviral restriction factors, whereas their protective properties are attributed at least in part to their production of antiviral interferon cytokines.
      Because the peritonea-derived, Fv3-resistant IL-34-Mϕs only exhibit greater expression of apobec2 than peritoneal CSF-1-Mϕs, this restriction factor may be particularly important to anti-Fv3 immunity. However, we emphasize that the differentiation of immune cells into distinct effectors results in lineage/subset-specific activation of unique and often non-overlapping gene regulatory networks (
      • Laslo P.
      • Pongubala J.M.
      • Lancki D.W.
      • Singh H.
      Gene regulatory networks directing myeloid and lymphoid cell fates within the immune system.
      ,
      • Singh H.
      • Khan A.A.
      • Dinner A.R.
      Gene regulatory networks in the immune system.
      ). In turn, it is this coordinated activation of specific repertoires of genes that grants those specialized cell types their respective functionally distinct capacities (
      • Laslo P.
      • Pongubala J.M.
      • Lancki D.W.
      • Singh H.
      Gene regulatory networks directing myeloid and lymphoid cell fates within the immune system.
      ,
      • Singh H.
      • Khan A.A.
      • Dinner A.R.
      Gene regulatory networks in the immune system.
      ). We postulate that the functional differences between CSF-1- and IL-34-Mϕs reflect their unique Mϕ subset “programing.” In turn, we propose that Mϕs differentiated by IL-34 are polarized toward a broad antiviral state through their expression of coordinated networks of genes related to antiviral defenses. Moreover, we suspect that the repertoires of these expressed genes far exceed those examined here, and the resistance of IL-34-Mϕs to Fv3 is conferred by potentially redundant or otherwise complementary mechanisms. This is supported by our observations that, while being highly resistant to Fv3 (
      • Grayfer L.
      • Robert J.
      Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages.
      ), frog peritonea-derived IL-34-Mϕs only express one of a subset of the (examined) restriction factors expressed by the bone marrow IL-34-Mϕs, as compared with the respective CSF-1-Mϕs. Moreover, we have previously shown that an elevated respiratory burst capacity of IL-34-Mϕs may also contribute to their Fv3 resistance (
      • Grayfer L.
      • Robert J.
      Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages.
      ). Frogs presumably possess multiple distinct subsets of IL-34-Mϕs, all of which are presumably resistant to Fv3, but as the result of at least partially non-overlapping antiviral mechanisms. The antiviral efficacies of these frog IL-34-Mϕ subsets may reflect their heightened production of and autocrine stimulation by IFNs, their expression of antiviral genes (such as restriction factors, IFN-stimulated genes), and their robust respiratory burst responses and more than likely result from other immunological parameters that are not considered here.
      Many aspects of Mϕ biology are interdependent on the activation of the colony-stimulating factor-1 receptor (
      • Lichanska A.M.
      • Browne C.M.
      • Henkel G.W.
      • Murphy K.M.
      • Ostrowski M.C.
      • McKercher S.R.
      • Maki R.A.
      • Hume D.A.
      Differentiation of the mononuclear phagocyte system during mouse embryogenesis: the role of transcription factor PU.1.
      ,
      • Guilbert L.J.
      • Stanley E.R.
      Specific interaction of murine colony-stimulating factor with mononuclear phagocytic cells.
      ). Considering the array of functionally disparate Mϕ subsets described across vertebrates, it is intuitive that some of this heterogeneity would stem from differential ligation of this receptor. CSF-1 was known to add to this Mϕ functional diversity (
      • Hamilton T.A.
      • Zhao C.
      • Pavicic Jr., P.G.
      • Datta S.
      Myeloid colony-stimulating factors as regulators of macrophage polarization.
      ), and the more recently discovered IL-34 cytokine appears to likewise aggrandize the Mϕ functional spectrum. The roles of IL-34 in vertebrate immunity appear to be complex and multifaceted, as this cytokine has been linked to the development of Langerhans cells (
      • Greter M.
      • Lelios I.
      • Pelczar P.
      • Hoeffel G.
      • Price J.
      • Leboeuf M.
      • Kündig T.M.
      • Frei K.
      • Ginhoux F.
      • Merad M.
      • Becher B.
      Stroma-derived interleukin-34 controls the development and maintenance of Langerhans cells and the maintenance of microglia.
      ,
      • Wang Y.
      • Colonna M.
      Interkeukin-34, a cytokine crucial for the differentiation and maintenance of tissue resident macrophages and Langerhans cells.
      ), osteoclasts (
      • Baud'huin M.
      • Renault R.
      • Charrier C.
      • Riet A.
      • Moreau A.
      • Brion R.
      • Gouin F.
      • Duplomb L.
      • Heymann D.
      Interleukin-34 is expressed by giant cell tumours of bone and plays a key role in RANKL-induced osteoclastogenesis.
      ,
      • Chen Z.
      • Buki K.
      • Vääräniemi J.
      • Gu G.
      • Väänänen H.K.
      The critical role of IL-34 in osteoclastogenesis.
      ), B cell–stimulating myeloid cells (
      • Yamane F.
      • Nishikawa Y.
      • Matsui K.
      • Asakura M.
      • Iwasaki E.
      • Watanabe K.
      • Tanimoto H.
      • Sano H.
      • Fujiwara Y.
      • Stanley E.R.
      • Kanayama N.
      • Mabbott N.A.
      • Magari M.
      • Ohmori H.
      CSF-1 receptor-mediated differentiation of a new type of monocytic cell with B cell-stimulating activity: its selective dependence on IL-34.
      ), and microglia (
      • Greter M.
      • Lelios I.
      • Pelczar P.
      • Hoeffel G.
      • Price J.
      • Leboeuf M.
      • Kündig T.M.
      • Frei K.
      • Ginhoux F.
      • Merad M.
      • Becher B.
      Stroma-derived interleukin-34 controls the development and maintenance of Langerhans cells and the maintenance of microglia.
      ). Moreover, IL-34 is produced by fibroblasts and synoviocytes of patients with rheumatoid arthritis (
      • Chemel M.
      • Le Goff B.
      • Brion R.
      • Cozic C.
      • Berreur M.
      • Amiaud J.
      • Bougras G.
      • Touchais S.
      • Blanchard F.
      • Heymann M.F.
      • Berthelot J.M.
      • Verrecchia F.
      • Heymann D.
      Interleukin 34 expression is associated with synovitis severity in rheumatoid arthritis patients.
      ), and its absence is correlated with reduced synaptic loss and less severe associated memory impairment in West Nile virus–infected mice (
      • Vasek M.J.
      • Garber C.
      • Dorsey D.
      • Durrant D.M.
      • Bollman B.
      • Soung A.
      • Yu J.
      • Perez-Torres C.
      • Frouin A.
      • Wilton D.K.
      • Funk K.
      • DeMasters B.K.
      • Jiang X.
      • Bowen J.R.
      • Mennerick S.
      • Robinson J.K.
      • Garbow J.R.
      • Tyler K.L.
      • Suthar M.S.
      • Schmidt R.E.
      • Stevens B.
      • Klein R.S.
      A complement-microglial axis drives synapse loss during virus-induced memory impairment.
      ), suggesting that IL-34-Mϕs have pro-inflammatory roles. The IL-34 produced during inflammatory responses has also been proposed to have anti-inflammatory roles (
      • Guillonneau C.
      • Bézie S.
      • Anegon I.
      Immunoregulatory properties of the cytokine IL-34.
      ). This is supported by the recent observation that IL-34 is produced by T regulatory cells and contributes to transplant tolerance (
      • Bézie S.
      • Picarda E.
      • Ossart J.
      • Tesson L.
      • Usal C.
      • Renaudin K.
      • Anegon I.
      • Guillonneau C.
      IL-34 is a Treg-specific cytokine and mediates transplant tolerance.
      ). Our work using the amphibian X. laevis-Fv3 infection model indicates that the frog IL-34 is crucial to the development of an antiviral Mϕ subset, and the absence of proper IL-34 production by amphibian tadpoles may be an important underlying cause of their greater susceptibility to this pathogen (
      • Grayfer L.
      • Robert J.
      Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34.
      ). It will be interesting to learn whether the antiviral properties of IL-34–derived Mϕs are conserved across vertebrate species or represent an amphibian phenomenon.
      Our results clearly indicate that IL-34-Mϕs are significantly more resistant to Fv3 infection and viral replication than CSF-1-Mϕs. We propose that a significant source of this resistance is the IL-34-Mϕ expression of antiviral restriction factors. Notably, cellular resistance to viral replication is marked by expression of antiviral restriction factors (
      • Badia R.
      • Angulo G.
      • Riveira-Muñoz E.
      • Pujantell M.
      • Puig T.
      • Ramirez C.
      • Torres-Torronteras J.
      • Marti R.
      • Pauls E.
      • Clotet B.
      • Ballana E.
      • Esté J.A.
      Inhibition of herpes simplex virus type 1 by the CDK6 inhibitor PD-0332991 (palbociclib) through the control of SAMHD1.
      • Ballana E.
      • Esté J.A.
      SAMHD1: at the crossroads of cell proliferation, immune responses, and virus restriction.
      ,
      • Lang F.
      • Li X.
      • Vladimirova O.
      • Hu B.
      • Chen G.
      • Xiao Y.
      • Singh V.
      • Lu D.
      • Li L.
      • Han H.
      • Wickramasinghe J.M.
      • Smith S.T.
      • Zheng C.
      • Li Q.
      • Lieberman P.M.
      • et al.
      CTCF interacts with the lytic HSV-1 genome to promote viral transcription.
      • Li D.J.
      • Verma D.
      • Mosbruger T.
      • Swaminathan S.
      CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription.
      ), such as those up-regulated by IL-34-Mϕs and including apobec (
      • Trang K.
      • Raposo R.A.
      • Lowe M.M.
      • Krow-Lucal E.R.
      • Yonemoto W.
      • Cabido V.D.
      • SenGupta D.
      • McCune J.M.
      Relative mRNA expression levels of restriction factors and antiviral genes in fetal and adult human monocytes and monocyte-derived macrophages.
      ) and trim (
      • Nisole S.
      • Stoye J.P.
      • Saïb A.
      TRIM family proteins: retroviral restriction and antiviral defence.
      ) family members, samhd1 (
      • Gramberg T.
      • Kahle T.
      • Bloch N.
      • Wittmann S.
      • Müllers E.
      • Daddacha W.
      • Hofmann H.
      • Kim B.
      • Lindemann D.
      • Landau N.R.
      Restriction of diverse retroviruses by SAMHD1.
      ), and ctcf (
      • Pentland I.
      • Parish J.L.
      Targeting CTCF to control virus gene expression: a common theme amongst diverse DNA viruses.
      ). Each of these gene products represents a barrier at a distinct step in viral replication, so the relative magnitudes of CSF-1- and IL-34-Mϕ expression of these genes probably contribute to their respective susceptibility and resistance to Fv3.
      It is noteworthy that whereas CSF-1-Mϕs supported greater expression of Fv3 IE, E, and L genes, these cells had particularly high expression of the Fv3 93L late gene compared with IL-34-Mϕs. Although the functional roles of the 93L gene product await elucidation, it is tempting to speculate that the resulting protein may have an important role in immune evasion and/or viral dissemination. It will be interesting to learn with further studies whether CSF-1-Mϕ susceptibility to Fv3 is somehow related to the elevated 93L gene expression within these cells.
      Our past work indicates that administration of IL-34 to tadpoles and adult frogs confers significant anti-Fv3 protection and reduces animal viral loads (
      • Grayfer L.
      • Robert J.
      Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34.
      ,
      • Grayfer L.
      • Robert J.
      Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages.
      ), whereas our present findings indicate that IL-34-Mϕ–conditioned supernatants possess factor(s) that render the frog A6 kidney epithelial line considerably more resistant to Fv3. Concomitantly, type I and type III IFNs are key mediators of vertebrate antiviral defenses (
      • Wang T.
      • Hanington P.C.
      • Belosevic M.
      • Secombes C.J.
      Two macrophage colony-stimulating factor genes exist in fish that differ in gene organization and are differentially expressed.
      ,
      • Hanington P.C.
      • Wang T.
      • Secombes C.J.
      • Belosevic M.
      Growth factors of lower vertebrates: characterization of goldfish (Carassius auratus L.) macrophage colony-stimulating factor-1.
      ,
      • Garceau V.
      • Smith J.
      • Paton I.R.
      • Davey M.
      • Fares M.A.
      • Sester D.P.
      • Burt D.W.
      • Hume D.A.
      Pivotal advance: avian colony-stimulating factor 1 (CSF-1), interleukin-34 (IL-34), and CSF-1 receptor genes and gene products.
      • Belosevic M.
      • Hanington P.C.
      • Barreda D.R.
      Development of goldfish macrophages in vitro.
      ), whereas amphibians encode highly expanded repertoires of phylogenetically distinct type I and type III IFNs (
      • Sang Y.
      • Liu Q.
      • Lee J.
      • Ma W.
      • McVey D.S.
      • Blecha F.
      Expansion of amphibian intronless interferons revises the paradigm for interferon evolution and functional diversity.
      ,
      • Gan Z.
      • Chen S.N.
      • Huang B.
      • Hou J.
      • Nie P.
      Intronless and intron-containing type I IFN genes coexist in amphibian Xenopus tropicalis: insights into the origin and evolution of type I IFNs in vertebrates.
      ). In this respect, it is particularly compelling that IL-34-Mϕs express significantly greater levels of type I and III IFN genes belonging to these diverse clades as well as their cognate receptors. Moreover, it appears that the A6 cell anti-Fv3 protection offered by IL-34–conditioned medium depends on IFN signaling, supporting our hypothesis that this Mϕ population offers antiviral protection by production of these antiviral cytokines. It will be interesting to learn the roles of the respective IFNs in amphibian antiviral immunity and the in vivo contexts in which IL-34-Mϕs produce these mediators.
      The lack of X. laevis CSF-1- and IL-34-Mϕ–specific markers has precluded the investigation of the roles of these cells in vivo. However, it is notable that IL-34 gene expression is higher than that of CSF-1 in the frog spleen tissues (
      • Grayfer L.
      • Robert J.
      Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34.
      ), suggesting that IL-34 may be more prominently involved in the frog innate and possibly adaptive immune defenses. The tadpoles of anuran amphibians are more susceptible to and die from Fv3, whereas adult frogs also suffer from productive infections but are usually able to clear these pathogens (
      • Grayfer L.
      • De Jesús Andino F.
      • Robert J.
      The amphibian (Xenopus laevis) type I interferon response to frog virus 3: new insight into ranavirus pathogenicity.
      ,
      • Bayley A.E.
      • Hill B.J.
      • Feist S.W.
      Susceptibility of the European common frog Rana temporaria to a panel of ranavirus isolates from fish and amphibian hosts.
      • Hoverman J.T.
      • Gray M.J.
      • Miller D.L.
      Anuran susceptibilities to ranaviruses: role of species identity, exposure route, and a novel virus isolate.
      ,
      • Landsberg J.H.
      • Kiryu Y.
      • Tabuchi M.
      • Waltzek T.B.
      • Enge K.M.
      • Reintjes-Tolen S.
      • Preston A.
      • Pessier A.P.
      Co-infection by alveolate parasites and frog virus 3-like ranavirus during an amphibian larval mortality event in Florida, U.S.A.
      • Reeve B.C.
      • Crespi E.J.
      • Whipps C.M.
      • Brunner J.L.
      Natural stressors and ranavirus susceptibility in larval wood frogs (Rana sylvatica).
      ). The tadpole and adult frog kidneys are a central site of Fv3 replication (
      • Morales H.D.
      • Robert J.
      Characterization of primary and memory CD8 T-cell responses against ranavirus (FV3) in Xenopus laevis.
      ,
      • Robert J.
      • Abramowitz L.
      • Gantress J.
      • Morales H.D.
      Xenopus laevis: a possible vector of Ranavirus infection?.
      ), and during FV3 infections, adult frogs up-regulate their kidney expression of both csf-1 and il-34 genes, whereas Fv3-challenged tadpoles increase their kidney csf-1 but not il-34 gene expression (
      • Grayfer L.
      • Robert J.
      Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34.
      ). Thus, the adult frogs presumably produce both the Fv3-susceptible CSF-1-Mϕs and the antiviral IL-34-Mϕ in response to viral challenge, whereas the failure of tadpoles to mount an IL-34 response and hence generate IL-34-Mϕs may contribute to their Fv3 susceptibility.
      Whereas Mϕs represent the foundation of vertebrate immunity, the mechanisms controlling the ontogeny of the many functionally disparate Mϕ subsets seen across vertebrata await elucidation. Presently, we demonstrate that CSF-1- and IL-34-Mϕ growth factors contribute to this functional spectrum by producing virally susceptible and resistant effector subsets. We believe that further insight into the functional dichotomies of CSF-1- and IL-34-Mϕs will not only permit greater understanding of the facets of amphibian susceptibility and resistance to ranaviruses, but will also grant a more comprehensive view of vertebrate Mϕ-pathogen interactions.

      Experimental procedures

      Animals

      Out-bred adult X. laevis were purchased from Xenopus1 and housed and handled under strict laboratory and institutional animal care and use committee regulations.

      Recombinant cytokines and cytokine receptors, A6 cell and Mϕ culture media, and conditions

      The production of recombinant X. laevis cytokines, isolation and culture of bone marrow Mϕs, and maintenance of A6 cells were performed as described previously (
      • Grayfer L.
      • Robert J.
      Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages.
      ,
      • Grayfer L.
      • De Jesús Andino F.
      • Robert J.
      Prominent amphibian (Xenopus laevis) tadpole type III interferon response to the frog virus 3 ranavirus.
      ,
      • Yaparla A.
      • Wendel E.S.
      • Grayfer L.
      The unique myelopoiesis strategy of the amphibian Xenopus laevis.
      ). The generation of recombinant extracellular domains of IFNAR2 and IFNLR1 was achieved using the same methodology described previously (
      • Grayfer L.
      • Robert J.
      Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages.
      ,
      • Grayfer L.
      • De Jesús Andino F.
      • Robert J.
      Prominent amphibian (Xenopus laevis) tadpole type III interferon response to the frog virus 3 ranavirus.
      ,
      • Yaparla A.
      • Wendel E.S.
      • Grayfer L.
      The unique myelopoiesis strategy of the amphibian Xenopus laevis.
      ). Bone-marrow IL-34- and CSF-1-Mϕ were generated by incubating 104 X. laevis bone marrow cells with 250 ng/ml recombinant CSF-1 or IL-34 in individual wells of 96-well plates for 5 days, with a second respective cytokine treatment added on the third day of culture. During reciprocal differentiation experiments, Mϕs were differentiated with CSF-1 or IL-34 as above and on the fifth day of culture, washed, and incubated with 250 ng/ml of the reciprocal cytokine for an additional 24 h. For A6 cell protection experiments, 104 A6 cells were incubated with CSF-1 or IL-34 Mϕs or supernatants derived from these cultures for 3 h before a 24-h Fv3 challenge (multiplicity of infection (MOI) = 0.5). Peritonea-derived IL-34- and CSF-1-Mϕs were generated as described previously (
      • Grayfer L.
      • Robert J.
      Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages.
      ).

      Morpholino targeting of IFNAR1 and IFNLR2

      Translation-blocking mo, specific for the X. laevis ifnar2 (mo 5′-CAGCAGAAGACACAGCCCTGCCATG-3′; targets both isoforms 2.1 and 2.2) and ifnlr1 (5′-GCCCAGGTGAACCAGACAGACATAT-3′) were designed by and purchased from GeneTools, LLC and introduced into A6 cells using the Endo-Porter PEG reagent according to the manufacturer’s directions (GeneTools, LLC).

      Quantitative gene expression analysis

      RNA and DNA isolation, cDNA synthesis, Mϕ and Fv3 gene expression, and Fv3 DNA load analyses were performed as described previously. All primers were validated before use, and all primer sequences are listed in Table S1.

      Statistical analysis

      All data were examined using analysis of variance and Student’s t test (Prism, GraphPad Software, La Jolla, CA); p < 0.05.

      Author contributions

      A.Y., M.P., and L.G. designed and performed all of the experiments and analyzed the results. A.Y. and L.G. wrote the manuscript and prepared the figures.

      References

        • Carey C.
        • Cohen N.
        • Rollins-Smith L.
        Amphibian declines: an immunological perspective.
        Dev. Comp. Immunol. 1999; 23 (10512457): 459-472
        • Daszak P.
        • Berger L.
        • Cunningham A.A.
        • Hyatt A.D.
        • Green D.E.
        • Speare R.
        Emerging infectious diseases and amphibian population declines.
        Emerg. Infect. Dis. 1999; 5 (10603206): 735-748
        • Grayfer L.
        • Andino Fde J.
        • Chen G.
        • Chinchar G.V.
        • Robert J.
        Immune evasion strategies of ranaviruses and innate immune responses to these emerging pathogens.
        Viruses. 2012; 4 (22852041): 1075-1092
        • Grayfer L.
        • Robert J.
        Amphibian macrophage development and antiviral defenses.
        Dev. Comp. Immunol. 2016; 58 (26705159): 60-67
        • Grayfer L.
        • Robert J.
        Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34.
        J. Leukoc. Biol. 2014; 96 (25190077): 1143-1153
        • Grayfer L.
        • Robert J.
        Distinct functional roles of amphibian (Xenopus laevis) colony-stimulating factor-1- and interleukin-34-derived macrophages.
        J. Leukoc. Biol. 2015; 98 (26136505): 641-649
        • Wang T.
        • Hanington P.C.
        • Belosevic M.
        • Secombes C.J.
        Two macrophage colony-stimulating factor genes exist in fish that differ in gene organization and are differentially expressed.
        J. Immunol. 2008; 181 (18714003): 3310-3322
        • Pixley F.J.
        • Stanley E.R.
        CSF-1 regulation of the wandering macrophage: complexity in action.
        Trends Cell Biol. 2004; 14 (15519852): 628-638
        • Hanington P.C.
        • Wang T.
        • Secombes C.J.
        • Belosevic M.
        Growth factors of lower vertebrates: characterization of goldfish (Carassius auratus L.) macrophage colony-stimulating factor-1.
        J. Biol. Chem. 2007; 282 (17827160): 31865-31872
        • Garceau V.
        • Smith J.
        • Paton I.R.
        • Davey M.
        • Fares M.A.
        • Sester D.P.
        • Burt D.W.
        • Hume D.A.
        Pivotal advance: avian colony-stimulating factor 1 (CSF-1), interleukin-34 (IL-34), and CSF-1 receptor genes and gene products.
        J. Leukoc. Biol. 2010; 87 (20051473): 753-764
        • Belosevic M.
        • Hanington P.C.
        • Barreda D.R.
        Development of goldfish macrophages in vitro.
        Fish Shellfish Immunol. 2006; 20 (15936214): 152-171
        • Droin N.
        • Solary E.
        Editorial: CSF1R, CSF-1, and IL-34, a “menage a trois” conserved across vertebrates.
        J. Leukoc. Biol. 2010; 87 (20430779): 745-747
        • Lin H.
        • Lee E.
        • Hestir K.
        • Leo C.
        • Huang M.
        • Bosch E.
        • Halenbeck R.
        • Wu G.
        • Zhou A.
        • Behrens D.
        • Hollenbaugh D.
        • Linnemann T.
        • Qin M.
        • Wong J.
        • Chu K.
        • et al.
        Discovery of a cytokine and its receptor by functional screening of the extracellular proteome.
        Science. 2008; 320 (18467591): 807-811
        • Wei S.
        • Nandi S.
        • Chitu V.
        • Yeung Y.G.
        • Yu W.
        • Huang M.
        • Williams L.T.
        • Lin H.
        • Stanley E.R.
        Functional overlap but differential expression of CSF-1 and IL-34 in their CSF-1 receptor-mediated regulation of myeloid cells.
        J. Leukoc. Biol. 2010; 88 (20504948): 495-505
        • Sadler A.J.
        • Williams B.R.
        Interferon-inducible antiviral effectors.
        Nat. Rev. Immunol. 2008; 8 (18575461): 559-568
        • Zou J.
        • Secombes C.J.
        Teleost fish interferons and their role in immunity.
        Dev. Comp. Immunol. 2011; 35 (21781984): 1376-1387
        • Sang Y.
        • Liu Q.
        • Lee J.
        • Ma W.
        • McVey D.S.
        • Blecha F.
        Expansion of amphibian intronless interferons revises the paradigm for interferon evolution and functional diversity.
        Sci. Rep. 2016; 6 (27356970): 29072
        • Gan Z.
        • Chen S.N.
        • Huang B.
        • Hou J.
        • Nie P.
        Intronless and intron-containing type I IFN genes coexist in amphibian Xenopus tropicalis: insights into the origin and evolution of type I IFNs in vertebrates.
        Dev. Comp. Immunol. 2017; 67 (27780747): 166-176
        • Grayfer L.
        • De Jesús Andino F.
        • Robert J.
        The amphibian (Xenopus laevis) type I interferon response to frog virus 3: new insight into ranavirus pathogenicity.
        J. Virol. 2014; 88 (24623410): 5766-5777
        • Grayfer L.
        • De Jesús Andino F.
        • Robert J.
        Prominent amphibian (Xenopus laevis) tadpole type III interferon response to the frog virus 3 ranavirus.
        J. Virol. 2015; 89 (25717104): 5072-5082
        • Wendel E.S.
        • Yaparla A.
        • Koubourli D.V.
        • Grayfer L.
        Amphibian (Xenopus laevis) tadpoles and adult frogs mount distinct interferon responses to the Frog Virus 3 ranavirus.
        Virology. 2017; 503 (28081430): 12-20
        • Shi K.
        • Carpenter M.A.
        • Banerjee S.
        • Shaban N.M.
        • Kurahashi K.
        • Salamango D.J.
        • McCann J.L.
        • Starrett G.J.
        • Duffy J.V.
        • Demir Ö.
        • Amaro R.E.
        • Harki D.A.
        • Harris R.S.
        • Aihara H.
        Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B.
        Nat. Struct. Mol. Biol. 2017; 24 (27991903): 131-139
        • Wolf D.
        • Hug K.
        • Goff S.P.
        TRIM28 mediates primer binding site-targeted silencing of Lys1,2 tRNA-utilizing retroviruses in embryonic cells.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (18713861): 12521-12526
        • Badia R.
        • Angulo G.
        • Riveira-Muñoz E.
        • Pujantell M.
        • Puig T.
        • Ramirez C.
        • Torres-Torronteras J.
        • Marti R.
        • Pauls E.
        • Clotet B.
        • Ballana E.
        • Esté J.A.
        Inhibition of herpes simplex virus type 1 by the CDK6 inhibitor PD-0332991 (palbociclib) through the control of SAMHD1.
        J. Antimicrob. Chemother. 2016; 71 (26542306): 387-394
        • Ballana E.
        • Esté J.A.
        SAMHD1: at the crossroads of cell proliferation, immune responses, and virus restriction.
        Trends Microbiol. 2015; 23 (26439297): 680-692
        • Lang F.
        • Li X.
        • Vladimirova O.
        • Hu B.
        • Chen G.
        • Xiao Y.
        • Singh V.
        • Lu D.
        • Li L.
        • Han H.
        • Wickramasinghe J.M.
        • Smith S.T.
        • Zheng C.
        • Li Q.
        • Lieberman P.M.
        • et al.
        CTCF interacts with the lytic HSV-1 genome to promote viral transcription.
        Sci. Rep. 2017; 7 (28045091): 39861
        • Li D.J.
        • Verma D.
        • Mosbruger T.
        • Swaminathan S.
        CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription.
        PLoS Pathog. 2014; 10 (24415941): e1003880
        • Dwyer A.R.
        • Mouchemore K.A.
        • Steer J.H.
        • Sunderland A.J.
        • Sampaio N.G.
        • Greenland E.L.
        • Joyce D.A.
        • Pixley F.J.
        Src family kinase expression and subcellular localization in macrophages: implications for their role in CSF-1-induced macrophage migration.
        J. Leukoc. Biol. 2016; 100 (26747837): 163-175
        • Grayfer L.
        • Hanington P.C.
        • Belosevic M.
        Macrophage colony-stimulating factor (CSF-1) induces pro-inflammatory gene expression and enhances antimicrobial responses of goldfish (Carassius auratus L.) macrophages.
        Fish Shellfish Immunol. 2009; 26 (19130890): 406-413
        • Donnelly R.P.
        • Kotenko S.V.
        Interferon-λ: a new addition to an old family.
        J. Interferon Cytokine Res. 2010; 30 (20712453): 555-564
        • Durbin R.K.
        • Kotenko S.V.
        • Durbin J.E.
        Interferon induction and function at the mucosal surface.
        Immunol. Rev. 2013; 255 (23947345): 25-39
        • Kotenko S.V.
        IFN-λs.
        Curr. Opin. Immunol. 2011; 23 (21840693): 583-590
        • Uzé G.
        • Monneron D.
        IL-28 and IL-29: newcomers to the interferon family.
        Biochimie. 2007; 89 (17367910): 729-734
        • Uzé G.
        • Schreiber G.
        • Piehler J.
        • Pellegrini S.
        The receptor of the type I interferon family.
        Curr. Top. Microbiol. Immunol. 2007; 316 (17969444): 71-95
        • Morales H.D.
        • Robert J.
        Characterization of primary and memory CD8 T-cell responses against ranavirus (FV3) in Xenopus laevis.
        J. Virol. 2007; 81 (17182687): 2240-2248
        • Robert J.
        • Abramowitz L.
        • Gantress J.
        • Morales H.D.
        Xenopus laevis: a possible vector of Ranavirus infection?.
        J. Wildl. Dis. 2007; 43 (17984259): 645-652
        • Laslo P.
        • Pongubala J.M.
        • Lancki D.W.
        • Singh H.
        Gene regulatory networks directing myeloid and lymphoid cell fates within the immune system.
        Semin. Immunol. 2008; 20 (18771937): 228-235
        • Singh H.
        • Khan A.A.
        • Dinner A.R.
        Gene regulatory networks in the immune system.
        Trends Immunol. 2014; 35 (24768519): 211-218
        • Lichanska A.M.
        • Browne C.M.
        • Henkel G.W.
        • Murphy K.M.
        • Ostrowski M.C.
        • McKercher S.R.
        • Maki R.A.
        • Hume D.A.
        Differentiation of the mononuclear phagocyte system during mouse embryogenesis: the role of transcription factor PU.1.
        Blood. 1999; 94 (10381505): 127-138
        • Guilbert L.J.
        • Stanley E.R.
        Specific interaction of murine colony-stimulating factor with mononuclear phagocytic cells.
        J. Cell Biol. 1980; 85 (6965942): 153-159
        • Hamilton T.A.
        • Zhao C.
        • Pavicic Jr., P.G.
        • Datta S.
        Myeloid colony-stimulating factors as regulators of macrophage polarization.
        Front. Immunol. 2014; 5 (25484881): 554
        • Greter M.
        • Lelios I.
        • Pelczar P.
        • Hoeffel G.
        • Price J.
        • Leboeuf M.
        • Kündig T.M.
        • Frei K.
        • Ginhoux F.
        • Merad M.
        • Becher B.
        Stroma-derived interleukin-34 controls the development and maintenance of Langerhans cells and the maintenance of microglia.
        Immunity. 2012; 37 (23177320): 1050-1060
        • Wang Y.
        • Colonna M.
        Interkeukin-34, a cytokine crucial for the differentiation and maintenance of tissue resident macrophages and Langerhans cells.
        Eur. J. Immunol. 2014; 44 (24737461): 1575-1581
        • Baud'huin M.
        • Renault R.
        • Charrier C.
        • Riet A.
        • Moreau A.
        • Brion R.
        • Gouin F.
        • Duplomb L.
        • Heymann D.
        Interleukin-34 is expressed by giant cell tumours of bone and plays a key role in RANKL-induced osteoclastogenesis.
        J. Pathol. 2010; 221 (20191615): 77-86
        • Chen Z.
        • Buki K.
        • Vääräniemi J.
        • Gu G.
        • Väänänen H.K.
        The critical role of IL-34 in osteoclastogenesis.
        PLoS One. 2011; 6 (21494622): e18689
        • Yamane F.
        • Nishikawa Y.
        • Matsui K.
        • Asakura M.
        • Iwasaki E.
        • Watanabe K.
        • Tanimoto H.
        • Sano H.
        • Fujiwara Y.
        • Stanley E.R.
        • Kanayama N.
        • Mabbott N.A.
        • Magari M.
        • Ohmori H.
        CSF-1 receptor-mediated differentiation of a new type of monocytic cell with B cell-stimulating activity: its selective dependence on IL-34.
        J. Leukoc. Biol. 2014; 95 (24052571): 19-31
        • Chemel M.
        • Le Goff B.
        • Brion R.
        • Cozic C.
        • Berreur M.
        • Amiaud J.
        • Bougras G.
        • Touchais S.
        • Blanchard F.
        • Heymann M.F.
        • Berthelot J.M.
        • Verrecchia F.
        • Heymann D.
        Interleukin 34 expression is associated with synovitis severity in rheumatoid arthritis patients.
        Ann. Rheum. Dis. 2012; 71 (22039170): 150-154
        • Vasek M.J.
        • Garber C.
        • Dorsey D.
        • Durrant D.M.
        • Bollman B.
        • Soung A.
        • Yu J.
        • Perez-Torres C.
        • Frouin A.
        • Wilton D.K.
        • Funk K.
        • DeMasters B.K.
        • Jiang X.
        • Bowen J.R.
        • Mennerick S.
        • Robinson J.K.
        • Garbow J.R.
        • Tyler K.L.
        • Suthar M.S.
        • Schmidt R.E.
        • Stevens B.
        • Klein R.S.
        A complement-microglial axis drives synapse loss during virus-induced memory impairment.
        Nature. 2016; 534 (27337340): 538-543
        • Guillonneau C.
        • Bézie S.
        • Anegon I.
        Immunoregulatory properties of the cytokine IL-34.
        Cell Mol Life Sci. 2017; 74 (28258292): 2569-2586
        • Bézie S.
        • Picarda E.
        • Ossart J.
        • Tesson L.
        • Usal C.
        • Renaudin K.
        • Anegon I.
        • Guillonneau C.
        IL-34 is a Treg-specific cytokine and mediates transplant tolerance.
        J. Clin. Invest. 2015; 125 (26389674): 3952-3964
        • Trang K.
        • Raposo R.A.
        • Lowe M.M.
        • Krow-Lucal E.R.
        • Yonemoto W.
        • Cabido V.D.
        • SenGupta D.
        • McCune J.M.
        Relative mRNA expression levels of restriction factors and antiviral genes in fetal and adult human monocytes and monocyte-derived macrophages.
        Viral Immunol. 2017; 30 (28151065): 142-148
        • Nisole S.
        • Stoye J.P.
        • Saïb A.
        TRIM family proteins: retroviral restriction and antiviral defence.
        Nat. Rev. Microbiol. 2005; 3 (16175175): 799-808
        • Gramberg T.
        • Kahle T.
        • Bloch N.
        • Wittmann S.
        • Müllers E.
        • Daddacha W.
        • Hofmann H.
        • Kim B.
        • Lindemann D.
        • Landau N.R.
        Restriction of diverse retroviruses by SAMHD1.
        Retrovirology. 2013; 10 (23497255): 26
        • Pentland I.
        • Parish J.L.
        Targeting CTCF to control virus gene expression: a common theme amongst diverse DNA viruses.
        Viruses. 2015; 7 (26154016): 3574-3585
        • Bayley A.E.
        • Hill B.J.
        • Feist S.W.
        Susceptibility of the European common frog Rana temporaria to a panel of ranavirus isolates from fish and amphibian hosts.
        Dis. Aquat. Organ. 2013; 103 (23574703): 171-183
        • Hoverman J.T.
        • Gray M.J.
        • Miller D.L.
        Anuran susceptibilities to ranaviruses: role of species identity, exposure route, and a novel virus isolate.
        Dis. Aquat. Organ. 2010; 89 (20402227): 97-107
        • Landsberg J.H.
        • Kiryu Y.
        • Tabuchi M.
        • Waltzek T.B.
        • Enge K.M.
        • Reintjes-Tolen S.
        • Preston A.
        • Pessier A.P.
        Co-infection by alveolate parasites and frog virus 3-like ranavirus during an amphibian larval mortality event in Florida, U.S.A.
        Dis. Aquat. Organ. 2013; 105 (23872853): 89-99
        • Reeve B.C.
        • Crespi E.J.
        • Whipps C.M.
        • Brunner J.L.
        Natural stressors and ranavirus susceptibility in larval wood frogs (Rana sylvatica).
        Ecohealth. 2013; 10 (23579812): 190-200
        • Yaparla A.
        • Wendel E.S.
        • Grayfer L.
        The unique myelopoiesis strategy of the amphibian Xenopus laevis.
        Dev. Comp. Immunol. 2016; 63 (27234705): 136-143