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Infections by ranaviruses such as Frog virus 3 (Fv3) are significantly contributing to the 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 re...
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... In this respect, amphibians represent a key stage in the evolution of vertebrate immunity and thus offer unique perspectives into the diverged and conserved aspects of vertebrate myelopoiesis [22,23]. Our ongoing studies using the Xenopus laevis frog model indicate that amphibian Mϕs differentiated by IL34 and CSF1 are morphologically, transcriptionally and functionally distinct [22,[24][25][26][27] and suggest that at least some of these differences have been evolutionarily conserved [28,29]. Notably, across our studies, we have noted that X. laevis IL34-Mϕs possess many features attributed to mammalian DCs [26,27]. ...
... Our ongoing studies using the Xenopus laevis frog model indicate that amphibian Mϕs differentiated by IL34 and CSF1 are morphologically, transcriptionally and functionally distinct [22,[24][25][26][27] and suggest that at least some of these differences have been evolutionarily conserved [28,29]. Notably, across our studies, we have noted that X. laevis IL34-Mϕs possess many features attributed to mammalian DCs [26,27]. To expand upon these observations, we produced a recombinant X. laevis FLT3L and compared the gene expression and functionality of the frog DCs generated by this factor to the X. laevis IL34-and CSF1-Mϕs. ...
... It is thus not surprising that the frog IL34-Mϕs share many features with the frog FLT3L-DCs. Our past work indicates that like mammalian plasmacytoid DCs [37], the X. laevis IL34-Mϕs also play important antiviral roles including the production of antiviral interferon cytokines [27] and express many of the surface markers, transcription factors and immune genes associated with mammalian DCs [26]. Indeed, in some inflammatory settings, monocytes give rise to monocyte-derived DCs (MoDCs) or inflammatory DCs [38], underscoring that even after lineage-commitment/differentiation, mammalian Mϕ-lineage cells retain the capacity to transition into DC (like) cells. ...
Macrophage (M ϕ )-lineage cells are integral to the immune defences of all vertebrates, including amphibians. Across vertebrates, M ϕ differentiation and functionality depend on activation of the colony stimulating factor-1 (CSF1) receptor by CSF1 and interluekin-34 (IL34) cytokines. Our findings to date indicate that amphibian ( Xenopus laevis ) M ϕ s differentiated with CSF1 and IL34 are morphologically, transcriptionally and functionally distinct. Notably, mammalian M ϕ s share common progenitor population(s) with dendritic cells (DCs), which rely on fms-like tyrosine kinase 3 ligand (FLT3L) for differentiation while X. laevis IL34-M ϕ s exhibit many features attributed to mammalian DCs. Presently, we compared X. laevis CSF1- and IL34-M ϕ s with FLT3L-derived X. laevis DCs. Our transcriptional and functional analyses indicated that indeed the frog IL34-M ϕ s and FLT3L-DCs possessed many commonalities over CSF1-M ϕ s, including transcriptional profiles and functional capacities. Compared to X. laevis CSF1-M ϕ s, the IL34-M ϕ s and FLT3L-DCs possess greater surface major histocompatibility complex (MHC) class I, but not MHC class II expression, were better at eliciting mixed leucocyte responses in vitro and generating in vivo re-exposure immune responses against Mycobacterium marinum . Further analyses of non-mammalian myelopoiesis akin to those described here, will grant unique perspectives into the evolutionarily retained and diverged pathways of M ϕ and DC functional differentiation.
This article is part of the theme issue ‘Amphibian immunity: stress, disease and ecoimmunology’.
... A6 cells have also been used to investigate the roles of secreted molecules from 552 CSF-1-or IL-34-differentiated X. laevis macrophages in cellular restriction to FV3. Following 553 infection, FV3 copy numbers were observed to be significantly lower in A6 cells pre-treated with 554 supernatants obtained from IL-34-differentiated macrophages than those from CSF-1-555 differentiated macrophages(Yaparla et al., 2018). This effect was reduced in A6 cells treated 556 with baricitinib, a Jak1/Tyk2 inhibitor, suggesting that IFNs were the causative factor.Transfection of A6 cells with antisense morpholinos targeting ifnar2.1/2.2 and ifnlr1 transcripts 558 and co-incubation with recombinant extracellular domains of IFNAR2.1 and IFNLR1 also 559 significantly reduced the effect, indicating that both type I and type III IFNs were contributing 560 (Yaparla et al., 2018). ...
Many amphibian populations are declining worldwide, and infectious diseases are a leading cause. Given the eminent threat infectious diseases pose to amphibian populations, there is a need to understand the host-pathogen-environment interactions that govern amphibian susceptibility to disease and mortality events. However, using animals in research raises an ethical dilemma, which is magnified by the alarming rates at which many amphibian populations are declining. Thus, in vitro study systems such as cell lines represent valuable tools for furthering our understanding of amphibian immune systems. In this review, we curate a list of the amphibian cell lines established to date (the amphibian invitrome), highlight how research using amphibian cell lines has advanced our understanding of the amphibian immune system, anti-ranaviral defence mechanisms, and Batrachochytrium dendrobatidis replication in host cells, and offer our perspective on how future use of amphibian cell lines can advance the field of amphibian immunology.
... We showed previously that frog bone marrow-derived macrophages differentiated with interleukin-34 (IL-34) but not with colony-stimulating factor 1 (CSF1; macrophage colony-stimulating factor [M-CSF]) are important producers of antiviral IFNs (34). Presently, we compared the gene expression of Xen1 in total tadpole kidney cells, the rCSF3-chemo-attracted kidney myeloid cells, and several X. laevis myeloid populations (Fig. 3F). ...
... Presumably, this ERV-mediated IFN augmentation has been co-opted into vertebrate immune systems. In support of this notion, it was interesting to see that adult X. laevis bone marrow-derived IL-34 macrophages, which are prominently involved in X. laevis antiviral IFN responses (34), also possessed significantly greater Xen1 expression than other bone marrow-derived cell types. Moreover, autocrine antiviral priming of cells through the constitutive production of antiviral IFNs is a well-accepted phenomenon (36)(37)(38) and it stands to reason that the production and immune recognition of ERV dsRNA may be a prominent mechanism by which this priming is maintained. ...
Global amphibian biodiversity is being challenged by pathogens like the Frog Virus 3 (FV3) ranavirus, underlining the need to gain a greater understanding of amphibian antiviral defenses. While it was previously believed that anuran (frog/toad) amphibian tadpoles are more susceptible to FV3, we demonstrated that tadpoles are in fact more resistant to this virus than metamorphic and postmetamorphic froglets.
... susceptible to FV3 infections [7]. Our recent work indicates the functional dichotomy of these frog MΦ subsets stems at least in part from their distinct pathogen recognition capacities [20,21] and from more robust antiviral type I and III interferon cytokine production by the IL-34-MΦs [22]. Conversely, the mechanisms through which the frog CSF-1-MΦs compromise the animals to FV3 have remained largely unexplored. ...
... CSF-1-and IL-34-MΦ enrichments affect a number of frog tissues, chiefly amongst them the frog kidneys [7,22,24], which are a central target for FV3 replication [23]. Accordingly, we next examined the kidneys of MΦ-enriched, FV3-infected animals to discern how these shifts in MΦ populations affect short-and long-term infection outcomes. ...
... Akin to previous studies that established that residual FV3 persists withing infected frog kidneys after the immune clearance of the primary infections [10,11], we also detected persisting FV3 DNA in the kidneys of frogs from all treatment groups, albeit in substantially lower levels at 21 and 28 dpi ( Figure 2B). them the frog kidneys [7,22,24], which are a central target for FV3 replication [23]. Accordingly, we next examined the kidneys of Mφ-enriched, FV3-infected animals to discern how these shifts in Mφ populations affect short-and long-term infection outcomes. ...
Infections by Frog Virus 3 (FV3) and other ranavirus genus members are significantly contributing to global amphibian decline. The Xenopus laevis frog is an ideal research platform upon which to study the roles of distinct frog leukocyte populations during FV3 infections. Frog macrophages (MΦs) are integrally involved during FV3 infection, as they facilitate viral dissemination and persistence but also participate in immune defense against this pathogen. In turn, MΦ differentiation and functionality depend on the colony-stimulating factor-1 receptor (CSF-1R), which is ligated by CSF-1 and iterleukin-34 (IL-34) cytokines. Our past work indicated that X. laevis CSF-1 and IL-34 give rise to morphologically and functionally distinct frog MΦ subsets, and that these CSF-1- and IL-34-MΦs respectively confer susceptibility and antiviral resistance to FV3. Because FV3 targets the frog kidneys and establishes chronic infections therein, presently we examined the roles of the frog CSF-1- and IL-34-MΦs in seeding and maintaining these chronic kidney infections. Our findings indicate that the frog CSF-1-MΦs result in more prominent kidney FV3 infections, which develop into greater reservoirs of lingering FV3 marked by infiltrating leukocytes, fibrosis, and overall immunosuppressive states. Moreover, the antiviral effects of IL-34-MΦs are short-lived and are lost as FV3 infections progress.
... We previously showed that certain X. laevis granulocyte subset(s) and macrophages differentiated by the interleukin-34 (IL-34) but not colony-stimulating factor-1 (CSF-1) macrophage growth factors, are important to these animals' antiviral defenses and exhibit broad ifn gene expression (18,(26)(27)(28). Notably, these granulocyte subset(s) and IL-34-macrophages, but not CSF-1macrophages, possess robust and specific esterase activity (18,19). ...
... It is notable that the FV3-infected tadpole intestinal ifn gene responses coincided with increased il34 and csf1 gene expression and the appearance of specific esterase-positive cells bearing IL-34-macrophage-like morphology. We previously demonstrated that X. laevis IL-34-macrophages, but not CSF-1-macrophages, possess robust specific esterase activity along with monocyte-like morphology and are important IFN producers (19,28,30). Notably, CSF-1 renders macrophages significantly more susceptible to FV3 (28), so the greater FV3 gene expression detected in the tadpole compared to the adult frog intestines may be stemming at least in part from an increased presence of CSF-1-macrophages. ...
... We previously demonstrated that X. laevis IL-34-macrophages, but not CSF-1-macrophages, possess robust specific esterase activity along with monocyte-like morphology and are important IFN producers (19,28,30). Notably, CSF-1 renders macrophages significantly more susceptible to FV3 (28), so the greater FV3 gene expression detected in the tadpole compared to the adult frog intestines may be stemming at least in part from an increased presence of CSF-1-macrophages. Alternatively, it is possible that both tadpole and adult frog intestinal cells are equally non-permissive to FV3 replication, and the decreased in vivo FV3-loads in tadpole intestines are due to greater presence of IL-34-macrophages, while the greater FV3 gene expression reflects the recruitment, expansion and/or polarization of CSF-1 macrophages at this site. ...
The global amphibian declines are compounded by ranavirus infections such as Frog Virus 3 (FV3), and amphibian tadpoles more frequently succumb to these pathogens than adult animals. Amphibian gastrointestinal tracts represent a major route of ranavirus entry, and viral pathogenesis often leads to hemorrhaging and necrosis within this tissue. Alas, the differences between tadpole and adult amphibian immune responses to intestinal ranavirus infections remain poorly defined. As interferon (IFN) cytokine responses represent a cornerstone of vertebrate antiviral immunity, it is pertinent that the tadpoles and adults of the anuran Xenopus laevis frog mount disparate IFN responses to FV3 infections. Presently, we compared the tadpole and adult X. laevis responses to intestinal FV3 infections. Our results indicate that FV3-challenged tadpoles mount more robust intestinal type I and III IFN responses than adult frogs. These tadpole antiviral responses appear to be mediated by myeloid cells, which are recruited into tadpole intestines in response to FV3 infections. Conversely, myeloid cells bearing similar cytology already reside within the intestines of healthy (uninfected) adult frogs, possibly accounting for some of the anti-FV3 resistance of these animals. Further insight into the differences between tadpole and adult frog responses to ranaviral infections is critical to understanding the facets of susceptibility and resistance to these pathogens.
... Interestingly, this has also been demonstrated in X. laevis infected with Frog virus 3 (Fv3) or Mycobacterium marinum that causes tuberculosis in aquatic vertebrates[186][187][188] . IL-34-treated macrophages possess more PRR at their surface compared to CSF-1-treated macrophages, leading to different downstream responses with greater production of type I and type III IFNs and antiviral restriction factors189,188 . These observations of PRR differential expression in X. laevis could be translated to humans and explain the differential role of IL-34-and CSF-1macrophages.Regarding enveloped RNA betacoronavirus, such as SARS-CoV-2, studies have shown the implication of pro-inflammatory monocytes/macrophages involved in a cytokine storm leading to severe acute respiratory distress syndrome and multiorgan pathology. ...
Although IL-34 and CSF-1 share actions as key mediators of monocytes/macrophages survival and differentiation, they also display differences that should be identified to better define their respective roles in health and diseases. IL-34 displays low sequence homology with CSF-1 but has a similar general structure and they both bind to a common receptor CSF-1R, although binding and subsequent intracellular signaling shows differences. CSF-1R expression has been until now mainly described at a steady state in monocytes/macrophages and myeloid dendritic cells, as well as in some cancers. IL-34 has also 2 other receptors, protein-tyrosine phosphatase zeta (PTPζ) and CD138 (Syndecan-1), expressed in some epithelium, cells of the central nervous system (CNS), as well as in numerous cancers. While most, if not all, of CSF-1 actions are mediated through monocyte/macrophages, IL-34 has also other potential actions through PTPζ and CD138. Additionally, IL-34 and CSF-1 are produced by different cells in different tissues. This review describes and discusses similarities and differences between IL-34 and CSF-1 at steady state and in pathological situations and identifies possible ways to target IL-34, CSF-1, and its receptors.
... properties than CSF1-activated macrophages. Similar to HIV-1 virus infection, FV3 infection induced the expression of the antiviral restriction factors IFNX, INOS and APOBEC in IL-34-activated macrophages [87]. Consequently, an increase in toll-like receptor 2 and 4 transcripts was detected in macrophages implicated in the recognition of bacterial cell wall LPS, as well as in the secretion of antiviral interferon IFN7 and tumor necrosis factor-alpha (TNF-α). ...
... Secretion of IL-34 by neurons in prion infections helps microglia proliferation and neuroprotection [20]. Moreover, IL-34 induces microglia resistance to West Nile virus infection and the same effects have been observed in other viral infections, such as HIV-1 [76] or FV3 [87]. In addition, IL-34 is associated with inducing and maintaining immune tolerance in liver transplantations, as well as during pregnancy [64]. ...
Macrophages are specialized cells that control tissue homeostasis. They include non-resident and tissue-resident macrophage populations which are characterized by the expression of particular cell surface markers and the secretion of molecules with a wide range of biological functions. The differentiation and polarization of macrophages relies on specific growth factors and their receptors. Macrophage-colony stimulating factor (CSF-1) and interleukine-34 (IL-34), also known as "twin" cytokines, are part of this regluatory landscape. CSF-1 and IL-34 share a common receptor, the macrophage-colony stimulating factor receptor (CSF-1R), which is activated in a similar way by both factors and turns on identical signaling pathways. However, there is some discrete differential activation leading to specific activities. In this review, we disscuss recent progress in understanding of the role of the twin cytokines in macrophage differentiation, from their interaction with CSF-1R and the activation of signaling pathways, to their implication in macrophage polarization of non-resident and tissue-resident macrophages. A special focus on IL-34, its involvement in pathophsyiological contexts, and its potential as a theranostic target for macrophage therapy will be proposed.
... As revealed in the current study, both intronless and intron-containing type I IFNs in N. parkeri were expressed at very low levels in unstimulated organs/tissues, but were highly induced by poly(I:C) stimulation, and they showed strong ability to induce ISGs and strong antiviral effect, as observed in most type I IFNs in mammals as well as in fish (1,3,13,20,62). However, previous research has shown that intron-containing type I IFNs in Xenopus were typical inducible cytokines with strong antiviral activity, whereas most intronless type I IFNs in Xenopus were constitutively expressed at basal expression level but were poorly inducible following poly(I:C) stimulation or virus infection and had very weak activity against virus infection, at least in A6 cells (21,22,26,63,64). Therefore, it seems possible that intronless and intron-containing type I IFNs in N. parkeri are functional with high degree of similarity in terms of the expression and bioactivity, but intronless and intron-containing type I IFNs in Xenopus may exhibit certain degree of difference in their expression and effect. ...
... *p , 0.05. effect (26,46,63,64). In the current study, the identified intronless type I IFNs, Np-IFNi1 and Np-IFNi2, in the Tibetan frog have been proven to have antiviral activity against FV3, implying that intronless type I IFN in amphibians may also play important roles in immune response against ranaviruses. ...
Type I interferons are a subset of cytokines playing central roles in host antiviral defense, and their effects depend on the interaction with the heterodimeric receptor complex. Surprisingly, two pairs of the receptor subunits, CRFB1 and CRFB5, and CRFB2 and CRFB5, have been identified in fish, but the studies about preferential receptor usage of different fish IFN subtypes are rather limited. In this study, the three receptor chains of type I IFNs named as On-CRFB1, On-CRFB2 and On-CRFB5 were identified in Nile tilapia, Oreochromis niloticus. These three genes were constitutively expressed in all tissues examined, with the highest expression level observed in muscle and liver, and were rapidly induced in liver following the stimulation of poly(I:C). Interestingly, it is possible that all three subtypes of tilapia IFNs are able to signal through two pairs of the receptor subunits, On-CRFB1 and On-CRFB5, and On-CRFB2 and On-CRFB5. More importantly, tilapia group I IFNs (On-IFNd and On-IFNh) preferentially signal through a receptor complex composed of On-CRFB1 and On-CRFB5, and group II IFNs (On-IFNc) preferentially signal through a receptor complex comprised of On-CRFB2 and On-CRFB5. The present study thus provides new insights into the receptor usage of group I and group II IFNs in fish.
... The production of recombinant rG-CSF, rCXCL8a, rCXCL8b and the recombinant control (rctrl) has been previously described Yaparla et al. 2018). In brief, the recombinant cytokines were produced by generating expression constructs containing the signal peptide-cleaved representations of the respective cytokine cDNAs in the pMIB/V5 His A insect expression vector (Invitrogen, Carlsbad, California, USA) backbone and transfecting these into Sf9 insect cells (Cellfectin II, Invitrogen). ...
... In brief, the recombinant cytokines were produced by generating expression constructs containing the signal peptide-cleaved representations of the respective cytokine cDNAs in the pMIB/V5 His A insect expression vector (Invitrogen, Carlsbad, California, USA) backbone and transfecting these into Sf9 insect cells (Cellfectin II, Invitrogen). Positive transfectants were scaled up to 500 ml cultures, grown for 5 days, and the recombinant proteins isolated from the supernatants of these cultures using Ni-NTA agarose (Qiagen, Hilden, Germany) columns as described previously Yaparla et al. 2018). The recombinant control (r-ctrl) was produced by transfecting Sf9 cells with an empty pMIB/V5 His A insect expression vector (Invitrogen) and processing the supernatants from the resulting transfected cultures using the same methodology as described for the above cytokines. ...
The ranavirus Frog Virus 3 (FV3) and the chytrid fungus Batrachochytrium dendrobatidis (Bd) are significant contributors to the global amphibian declines and both pathogens target the amphibian skin. We previously showed that tadpoles and adults of the anuran amphibian Xenopus laevis express notable levels of granulocyte chemokine genes (cxcl8a and cxcl8b) within their skin and likely possess skin-resident granulocytes. Presently, we show that tadpole and adult X. laevis indeed possess granulocyte-lineage cells within their epidermises that are distinct from their skin mast cells, which are found predominantly in lower dermal layers. These esterase-positive cells responded to (r)CXCL8a and rCXCL8b in a concentration- and CXCR1/CXCR2-dependent manner, possessed polymorphonuclear granulocyte morphology, granulocyte marker surface staining, and exhibited distinct immune gene expression from conventional granulocytes. Our past work indicates that CXCL8b recruits immunosuppressive granulocytes, and here we demonstrated that enriching esterase-positive skin granulocytes with rCXCL8b (but not rCXCL8a) may increase tadpole susceptibility to FV3 and adult frog susceptibility to Bd. Furthermore, pharmacological depletion of skin-resident granulocytes increased tadpole susceptibility to FV3. This manuscript provides new insights into the composition and roles of immune cells within the amphibian skin, which is a critical barrier against pathogenic contributors to the amphibian declines.
... Based on work using the relatively less FV3-suceptible Xenopus laevis frog model, it has been shown that frog macrophages (Mφs) are crucial to both the FV3 infection strategy as well as the immune defenses against this pathogen [5,6]. Interestingly, X. laevis Mφs differentiated with either colony-stimulating factor 1 (CSF-1) or interleukin-34 (IL-34) Mφ growth factors are highly susceptible and resistant to FV3, respectively [7]. FV3, and presumably at least some of the other iridoviruses, are infectious as both enveloped and naked virions [8][9][10]. ...
... The BM cells were flushed out of the femur bones with ice-cold APBS, collected, and washed with ice-cold APBS. Freshly isolated BM cells (10 5 cells) were enumerated and cultured with recombinant CSF-1 or IL-34 (250 ng/mL; produced as previously described [7]) for 5 days prior to the SR-A ligands/FV3 experiments. ...
... The effects of SR-A competitive and non-competitive ligands on blocking FV3 during the viral entry process were also studied in the X. laevis FV3-susceptible CSF-1-Mφs and the FV3-resistant IL-34-Mφs [7]. Notably, these Mφ populations are equally permissive to viral entry, while the IL-34-Mφ subset is more effective at eliminating the invading virus [5,6]. ...
Frog virus 3 (FV3) is the type species of the genus Ranavirus (family Iridoviridae). FV3 and FV3-like viruses are globally distributed infectious agents with the capacity to replicate in three vertebrate classes (teleosts, amphibians, and reptiles). At the cellular level, FV3 and FV3-like viruses can infect cells from virtually all vertebrate classes. To date, the cellular receptors that are involved in the FV3 entry process are unknown. Class A scavenger receptors (SR-As) are a family of evolutionarily conserved cell-surface receptors that bind a wide range of chemically distinct polyanionic ligands and can function as cellular receptors for other DNA viruses, including vaccinia virus and herpes simplex virus. The present study aimed to determine whether SR-As are involved in FV3 cellular entry. By using well-defined SR-A competitive and non-competitive ligand-blocking assays and absolute qPCR, we demonstrated that the SR-A competitive ligands drastically reduced the quantities of cell-associated viral loads in frog cells. Moreover, inducing the expression of a human SR-AI in an SR-A null cell line significantly increased FV3–cell association. Together, our results indicate that SR-As are utilized by FV3 during the cellular entry process.
