Endoplasmic Stress Affects the Coinfection of Leishmania Amazonensis and the Phlebovirus (Bunyaviridae) Icoaraci

Viral coinfections can modulate the severity of parasitic diseases, such as human cutaneous leishmaniasis. Leishmania parasites infect thousands of people worldwide and cause from single cutaneous self-healing lesions to massive mucosal destructive lesions. The transmission to vertebrates requires the bite of Phlebotomine sandflies, which can also transmit Phlebovirus. We have demonstrated that Leishmania infection requires and triggers the Endoplasmic stress (ER stress) response in infected macrophages. In the present paper, we tested the hypothesis that ER stress is increased and required for the aggravation of Leishmania infection due to coinfection with Phlebovirus. We demonstrated that Phlebovirus Icoaraci induces the ER stress program in macrophages mediated by the branches IRE/XBP1 and PERK/ATF4. The coinfection with L. amazonensis potentiates and sustains the ER stress, and the inhibition of IRE1α or PERK results in poor viral replication and decreased parasite load in macrophages. Importantly, we observed an increase in viral replication during the coinfection with Leishmania. Our results demonstrated the role of ER stress branches IRE1/XBP1 and PERK/ATF4 in the synergic effect on the Leishmania increased load during Phlebovirus coinfection and suggests that Leishmania infection can also increase the replication of Phlebovirus in macrophages.


Introduction
Cells may face stress conditions due to various sources of extracellular or intracellular factors. The cellular stress response is an adaptive process to reestablish cell homeostasis. The Endoplasmic Reticulum (ER) houses different processes, such as protein folding, lipid synthesis, and the biogenesis of autophagosomes and peroxisomes [1,2]. Whenever a stress condition interferes with protein synthesis or folding, ER initiates transcription and translational response, which involves lipid synthesis, the upregulation of chaperone expression, and the coordination of the retrograde transport and degradation of unfolded proteins (ERAD), [3].
The ER stress response comprises three branches: the transcription factor 6 (ATF6), inositol-requiring kinase 1 (IRE-1), and the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). These arms of ER stress initiate with the engagement of ER sensors and culminate with the activation of a set of transcription factors [4]. The sensor IRE-1 activates the X-box binding protein 1 (XBP-1) due to the cytoplasmic splicing of the X-box binding protein 1 (XBP-1) RNA, leading to the translation of the mature XBP-1 RNA [5,6]. The protein kinase R (PKR)-like (PERK) phosphorylates the initiation translational factor eIF2α, which restricts the overall protein translation and upregulates the expression of activating transcription factor 4 (ATF4) [7,8]. The reactivated translation of ATF-4-dependent genes may generate significant amounts of reactive oxygen species (ROS), leading to cell death.
Leishmaniasis is an emerging parasitic disease caused by several species of Leishmania and affects~12 million people worldwide, with approximately 2 million new infections per year [14]. Depending on the species and the host immune response, diverse clinical manifestations can be observed ranging from cutaneous to visceral form of the infection. Leishmania parasites reach the vertebrate hosts through the regurgitation and inoculation of metacyclic promastigotes by Phlebotomine sandfly vectors. Inside the vertebrate hosts, promastigotes replicate as amastigotes in macrophages, subverting the immune response [15,16].
Leishmania amazonensis is endemic in Brazil and is the causative agent of local cutaneous leishmaniasis (LCL) and a more severe form, the anergic diffuse cutaneous leishmaniasis (ADCL). The immunological response is associated to a T-cell hyposensitivity pole and patients present a nonreactive profile characterized by negative delayed type hypersensitivity (DTH), [17] We demonstrated that Leishmania amazonensis promotes the ER stress response in infected macrophages, and immunohistological analysis of human clinical samples showed the expression of XBP-1 and ATF4 [18,19]. Another group reported similar results in L. infantum infected macrophages [20]. The Amazon Region presents different Leishmania species with distinct sylvatic cycles, covering a diversity of sandflies vectors and natural reservoirs. L amazonensis distribution was initially described in some regions of the North of Brazil. However, autochthonous transmission may take place in other regions, including periurban areas [21].
The Brazilian Amazon region is probably the largest arboviruses reserve in the world [22], which may favor coinfections due to the convergence of hosts and vectors shared by Leishmania and arbovirus transmitted by sandflies.
Leishmania vectors can also transmit arbovirus belonging to the Family Bunyaviridae, and they are known as Phlebovirus [23]. Phlebovirus are now in the Phenuiviridae family, the term Bunyaviridae has been substituted with Phenuiviridae [24,25], causing distinct symptoms [26,27]. Some studies showed overlapped phleboviruses and Leishmania transmission areas, and sand fly coinfections were reported [28,29]. Our previous studies utilizing in vivo and in vitro models demonstrated that an Amazonian Phlebovírus (Icoaraci, ICOV) which shares the same vector and wild reservoir of L. amazonensis, potentiated the parasite infection [30].
The present paper reports that ICOV induces and enhances a sustained ER stress response in coinfection with L. amazonensis, which is partially required for the aggravation of the parasitic infection. Importantly, ICOV replication is also favored in the coinfection of macrophages with Leishmania, thus suggesting a mutual benefit of the pathogens inside vertebrate cells.

Peritoneal Macrophages
Thioglycolate-elicited peritoneal macrophages from eight-week-old wild-type (WT) C57BL/6 mice were elicited with 2 mL of 3% sodium thioglycolate Brewer solution for 4 days and obtained by injecting PBS into the peritoneal cavity. The cell suspension was washed in PBS once and then resuspended in serum-free DMEM. Cells were plated on glass coverslips at 2 × 105/well in 24-well polystyrene plates or 4 × 106/well in 6-well polystyrene plates and incubated for 1 h at 37 • C in a 5% CO 2 atmosphere. Nonadherent cells were washed out with PBS, and the adherent cell population was incubated overnight in DMEM with high glucose and supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 g/mL streptomycin for subsequent treatment or Leishmania/virus infection assays.

Phlebovirus: Production, Quantification, and Infection
The Phlebovirus Icoaraci (BeAN 24262-ICOV) used in this work were obtained through Dr. Pedro Vasconcelos (Instituto Evandro Chagas/SVS/MS). Viruses from lyophilized mouse brain macerates were passaged five times in BHK-21 cells to recover infectivity. For viral propagation, BHK-21 cells were seeded in 100-mm plates and the virus inoculum was adsorbed for 2 h at room temperature with a MOI (multiplicity of infection) of 0.01. The inoculum was removed and the cells were maintained in DMEM with high glucose and supplemented with 10% heat-inactivated FBS at 37 • C for 3 days. For viral concentration, cell culture supernatants containing viral particles were collected at 18,000× g for 1 h at 4 • C. The pellet was reconstituted in PBS (Gibco) and stored at −80 • C. Viral titers were determined by plaque assay. BHK-21 cells were seeded in 6-well polystyrene plates (initial inoculum of 1.5 × 10 6 cells/well). After 24 h, the cells were infected with a 10-fold of serial dilution of ICOV stock and incubated for 2 h at room temperature. Subsequently, the inoculum was removed and the monolayer covered with semisolid medium composed of DMEM with high glucose supplemented with 10% FBS and 1% methylcellulose (Sigma-Aldrich, Saint Louis, MO, USA). Viral plaques were observed 3 days after infection when cells were fixed and stained with a 0.1% crystal violet solution containing 10% formaldehyde. Viral plaques were counted and titers were expressed as PFU/mL. For macrophage infections, adsorption was performed for 1 h at 37 • C using a MOI of 1/1. Infection times varied according to the objective of the experiment, [30,31].

Parasites, Culture Conditions, and Infection
L. (L) amazonensis (WHOM/R/75/Josefa) was maintained in vitro in Schneider's Insect Medium (Sigma) supplemented with 10% of fetal bovine serum. The infection was carried out with stationary phase promastigotes at a parasite-to-cell ratio of 5:1 in triplicate. For the survival assays, peritoneal macrophages were infected for 48 h at 35 • C, followed by washing, fixation with methanol, and Giemsa staining. Infected macrophages were counted by light microscopy to assess the infection index as follows: 100 Giemsa-stained cells were inspected and the percentage of infected macrophages was multiplied by the average number of amastigotes per macrophage. Macrophages were treated with 40 nM of the PERK inhibitor (cat GSK2606414-MERCK.) or 100 nM IRE1α inhibitor (cat 4µ8c 14003-96-4 MERK).
After ICOV adsorption, the cultures were washed with PBS, and the macrophages were infected with promastigotes at the stationary phase with a ratio of 5:1.

En Bloc Processing for Transmission Electron Microscopy (TEM)
For TEM analysis, mock and infected macrophages in 25 cm 2 plastic culture flasks were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and post-fixed for 1 h in 1% OsO 4 0.8% potassium ferrocyanide in the same buffer. Then, samples were incubated Viruses 2022, 14,1948 4 of 13 with 2.5% uranyl acetate in water for 2 h, washed three times in 0.1 M cacodylate buffer (pH 7.2), dehydrated in ethanol, and embedded in Polybed resin (Polysciences). Spatial orientation of the samples was maintained for ultrathin sections, which were stained with 5% uranyl acetate (40 min) and 4% lead citrate (5 min) before observation using Hitachi HT7800 transmission electron microscope equipped with an AMT CMOS 8K camera.

Statistical Analysis
Data were analyzed by two-way ANOVA for independent samples followed by a post hoc Bufferoni compare all columns (with no designated control group), using GraphPad Prism 5 software (San Diego, CA, USA). Data were presented as the mean values ± standard error of the mean (SEM) of three independent experiments. Comparisons between means were considered to be statistically significant when p < 0.05.

Ultrastructure Aspects of ICOV in Murine Macrophages
We pursued TEM experiments in macrophages infected by ICOV to get insights into the interaction with macrophages, which are primary cells infected by Leishmania.
Macrophages infected with ICOV (MOI = 1) were processed at 24 h post-infection for visualization by transmission electron microscopy (TEM). At this time, viral particles were observed attached to the cell surface ( Figure 1A,B). However, ICOV seemed to be also present in the lumen of large vacuoles ( Figure 1C,D). In another macrophage, the interaction between the virus and the inner face of the vacuolar membrane displayed a belt-like structure, suggesting the virus pinch-off that allows the virus to occupy the lumen of the vacuole. Macrophages infected with ICOV (MOI = 1) were processed at 24 h post-infection for visualization by transmission electron microscopy (TEM). At this time, viral particles were observed attached to the cell surface ( Figure 1A,B). However, ICOV seemed to be also present in the lumen of large vacuoles ( Figure 1C,D). In another macrophage, the interaction between the virus and the inner face of the vacuolar membrane displayed a belt-like structure, suggesting the virus pinch-off that allows the virus to occupy the lumen of the vacuole. ICOV was also observed in canyons adjacent to cell membrane protrusions ( Figure 1E,F), a site of extensive actin activity. Discontinuation of cell plasma membrane indicates the viral egress. In the same image, the en bloc contrasting that makes the viral nucleic acid more Viruses 2022, 14,1948 6 of 13 electrondense for visualization by TEM revealed a virus-like-particle, tightly adhered to the cell surface.

ICOV Enhances Leishmania Growth through the ER Stress Response
Viral infections induce and may require ER the stress response to replicate inside infected cells [32,33]. We decided to test whether the inhibition of IRE1α or PERK would affect the exacerbated parasitic growth due to ICOV coinfection. Figure 2A,B confirms our previous findings on increasing the L. amazonensis load and the number of infected macrophages upon viral coinfection, which is comparable with the induction of ER stress by thapsigargin. Then, we tested the role of IRE1α in the context of coinfection. Figure 2C,D shows the reduction of the parasite load by the inhibitor, even in the presence of ICOV. We decided to address whether PERK/ATF4 would play a role in the coinfection. Figure 1E,F shows the reduction of the parasite load and the percentual of infected macrophages 48 h post coinfection due to the inhibition of PERK. Macrophage and Leishmania viability were not affected during treatment with IRE1α and PERK inhibitors We decided to address whether PERK/ATF4 would play a role in the coinfection. Figure 1E,F shows the reduction of the parasite load and the percentual of infected macrophages 48 h post coinfection due to the inhibition of PERK. Macrophage and Leishmania viability were not affected during treatment with IRE1α and PERK inhibitors ( Figure S1A,B).
We concluded that two branches of the ER response, IRE1α and PERK/ATF4, are necessary to enhance parasitic growth in the ICOV coinfection model.

The ER Stress Branch IRE1α/XBP1 Is Required for ICOV Replication, and It Is Augmented and Sustained during Coinfection
IRE1a links the ER to the nucleus by modulating the XBP-1 activity and the expression of several genes involved in the Unfolded Protein Response (UPR). IRE1 alpha protein possesses intrinsic kinase and endoribonuclease activity; the latter is responsible for the splicing of unprocessed uXBP1 to sXBP1 mRNA, which will be translated to the active XBP1 transcription factor [34].
We sought to investigate if ICOV induces IRE1a/XBP1 signaling during the infection. Figure 3A shows the expression of sXBP1 in ICOV infected macrophages. Then, we tested whether the coinfection by ICOV/L.amazonensis would affect the levels of sXBP1mRNA. Interestingly, the coinfection leads to increased levels of sXBP1 mRNA within 6 h of infection ( Figure 3B protein possesses intrinsic kinase and endoribonuclease activity; the latter is responsible for the splicing of unprocessed uXBP1 to sXBP1 mRNA, which will be translated to the active XBP1 transcription factor [34]. We sought to investigate if ICOV induces IRE1a/XBP1 signaling during the infection. Figure 3A shows the expression of sXBP1 in ICOV infected macrophages. Then, we tested whether the coinfection by ICOV/L.amazonensis would affect the levels of sXBP1mRNA. Interestingly, the coinfection leads to increased levels of sXBP1 mRNA within 6 h of infection ( Figure 3B   We decided to test whether the coinfection would benefit viral replication. Figure 3C shows the increase in the number of viral particles measured by plaque assays in the coinfection condition. Figure 3D shows the rise in the mRNA for the structural viral protein N during coinfection, thus supporting the viral plaque results. Notably, the chemical inhibition of IRE1alfa diminished viral replication.
These results suggest the role of IRE1alpha/XBP1 in the replication of ICOV and in favoring Leishmania growth during coinfection.

ICOV/Leishmania Coinfection Increases the Expression of XBP1-Regulated Genes Involved in the Synthesis of Phosphatidylcholine
The expansion of the ER demands phospholipids, such as phosphocholine, which is abundant in mammalian cell membranes [35]. Since XBP1 regulates a set of genes involved in the phospholipid synthesis, we decided to address whether the ER stress-induced during the infection by ICOV and L. amazonensis would upregulate the expression of the cholinephosphotransferases CPT1 and CEPT1enzymes, which regulate the final steps of the Kennedy pathways [36]. Figure 4A,B shows the augmented mRNA levels of CHPT1 during ICOV infection and upon the coinfection, respectively. Notably, the coinfection resulted in a slight but significant increase in the expression compared to the single infection by L. amazonensis or ICOV. In Figure 4C,D, we observed a similar mRNA expression profile for CEPT1. Significantly, the chemical inhibition of IRE1α abolished the expression of CHP1 and CEPT1. The enhanced expression of CHPT1 and CEPT1 is sustained for 24 h after coinfection ( Figure S3A-D).

ICOV/Leishmania Coinfection Increases the Expression of XBP1-Regulated Genes Involved in the Synthesis of Phosphatidylcholine
The expansion of the ER demands phospholipids, such as phosphocholine, which is abundant in mammalian cell membranes [35]. Since XBP1 regulates a set of genes involved in the phospholipid synthesis, we decided to address whether the ER stressinduced during the infection by ICOV and L. amazonensis would upregulate the expression of the cholinephosphotransferases CPT1 and CEPT1enzymes, which regulate the final steps of the Kennedy pathways [36]. Figure 4A,B shows the augmented mRNA levels of CHPT1 during ICOV infection and upon the coinfection, respectively. Notably, the coinfection resulted in a slight but significant increase in the expression compared to the single infection by L. amazonensis or ICOV. In Figure 4C,D, we observed a similar mRNA expression profile for CEPT1. Significantly, the chemical inhibition of IRE1α abolished the expression of CHP1 and CEPT1. The enhanced expression of CHPT1 and CEPT1 is sustained for 24 h after coinfection ( Figure S3A-D).  coinfection (B,D). Asterisks indicate significant differences with ** p < 0.001 or *** p < 0.0001.
We concluded that XBP1 is transcriptionally active in our models and may regulate the expression of genes involved in ER membrane expansion.

ICOV and the Coinfection with L. amazonensis Engage the ER Stress Branch PERK/ATF4.
Then, we addressed if ICOV and the coinfection with L. amazonensis would increase the mRNA levels of ATF4 and NRF2, regulated by the ER stress sensor PERK. Figure 5A,B, respectively, shows the ICOV upregulation of ATF4 and enhanced levels during the  coinfection (B,D). Asterisks indicate significant differences with ** p < 0.001 or *** p < 0.0001.
We concluded that XBP1 is transcriptionally active in our models and may regulate the expression of genes involved in ER membrane expansion.

ICOV and the Coinfection with L. amazonensis Engage the ER Stress Branch PERK/ATF4
Then, we addressed if ICOV and the coinfection with L. amazonensis would increase the mRNA levels of ATF4 and NRF2, regulated by the ER stress sensor PERK. Figure 5A,B, respectively, shows the ICOV upregulation of ATF4 and enhanced levels during the coinfection. NRF2 is a master transcription factor that regulates the expression of genes involved in the antioxidative stress response [37], induced via the PERK/ATF4 pathway. Figure 5C,D shows the upregulation of NRF2 by ICOV expression due to the coinfection. The pharmacological inhibition of PERK diminished the mRNA levels of ATF4 and NRF2, thus demonstrating the dependence of PERK activation. We observed a significant increase in ATF4 and NRF2 mRNA levels during the coinfection, which is sustained for 24 h, suggesting the potentiation of the antioxidative response in this condition. (Figure S4A-D).
Viruses 2022, 14, x FOR PEER REVIEW 12 of 16 Figure 5C,D shows the upregulation of NRF2 by ICOV expression due to the coinfection. The pharmacological inhibition of PERK diminished the mRNA levels of ATF4 and NRF2, thus demonstrating the dependence of PERK activation. We observed a significant increase in ATF4 and NRF2 mRNA levels during the coinfection, which is sustained for 24 h, suggesting the potentiation of the antioxidative response in this condition. ( Figure  S4A-D).

Discussion
Phleboviruses comprise a group of three segmented RNA viruses transmitted to vertebrates by Phlebotomine sandflies and other arthropods, such as ticks [38]. Approximately 67 phleboviruses species are recognized, and they share a high degree of amino acid identity in the RNA-dependent RNA polymerase coded in the L RNA segment genome [23].
Phlebotominae species are the primary vectors of Leishmania, which are obligatory intracellular parasites in vertebrates, including humans. The human infection by Leishmania species may cause a plethora of symptoms, varying from limited self-healing cutaneous ulcers and deforming mucosal lesions to lethal visceral conditions [39]. Transmission of cutaneous Leishmaniasis and phleboviruses overlap geographically [40], and molecular PCR tests revealed the coinfection of a limited set of Phlebotomus in Europe [41]. A growing body of in vitro and in vivo evidence suggests the potential importance of Phleboviruses in the aggravation of Leishmaniasis and the enhancement of parasite replication [30,42,43].
Our TEM studies revealed some aspects of the interaction of ICOV with macrophages, which are the primary cell type infected by Leishmania parasites. We could not attest that the viral particles shown in Figure 1C,D were within a large vacuole since the image refers to a 60 µm slice of the sample. In the same way, the belt-like structure observed in Figure 1E suggests a disconnection in progress between both. However, if what we noticed as a vacuole was, indeed, part of the macrophage plasma membrane projection, the phenomenon could result from the initial mechanisms that lead to virus internalization. Future tomography analysis of similar virus-cell interactions may resolve these uncertainties. The presence of Phleboviruses in canyons at the base of cell membrane projections, in sites where the plasma membrane is discontinued ( Figure 1F), is suggestive of viral egress.
ICOV was isolated from the rodent Nectomys squamipes, which may serve as an L. (L.) amazonensis reservoir, and it shares the same environment as the vector Lutzomyia flaviscutellata, the primary vector of L. amazonensis. Some reports suggest that ICOV may occur in a broad geographical distribution in Brazil, and serological studies revealed the occurrence of ICOV in distinct vertebrates, such as ruminants and non-human primates [44].
Viruses may induce the ER stress response to adapt the host cell machinery to produce new synthesized viral particles [13,32]. Under viral infection, some branches of ER stress are activated, reprogramming the transcriptional and translational programs [32]. The expression of genes involved in synthesizing the phospholipids under XBP-1 control and the transcription of antioxidative response genes mediated by the activation of NRF2 is a common feature of the induced ER stress program.
To fully ascertain that ER stress is occurring in our models and the majority of the literature, measuring the mass of unfolded or misfolded proteins inside the ER would be necessary. We did not address this point. However, we could establish the engagement of ER stress sensors, PERK and IRE1, in addition to the processing of XBP1 and the expression of some genes regulated in response to the ER stress response. Our data support the notion that ICOV and Leishmania infection cooperatively mobilize the ER stress program. We demonstrated that ICOV induces the XBP-1 and PERK/ATF4 mediated arms of ER stress, and the pharmacological inhibition of IRE1 or PERK reduces the viral replication in macrophages. The coinfection with L.amazonensis potentiated and sustained the ER stress response, apparently leading to a mutual benefit in viral and parasite L. amazonensis infections may be limited in humans, causing localized cutaneous lesions. More rarely, this species is found in patients with the anergic diffuse cutaneous leishmaniasis (ADCL), with a poor TH1 response and abundant amastigote forms [45]. Previously, we showed increased levels of the dsRNA sensor PKR and IFNb in the lesions of ADCL patients [46,47]. It is attempting to hypothesize that viral RNA coinfection may be implicated in the outcome of the infection. The coinfection in vivo assays demonstrated with ICOV (28) revealed high levels of IFNβ and IL-10, which may partially mirror the finding in some clinical studies [45]. Our findings suggest that the ER stress response is one component that may shape the parasite's replication inside macrophages. Currently, we are addressing the coinfection in cutaneous leishmaniasis and the expression of ER stress markers' survival and growth inside the host cell. This finding might be of epidemiological importance considering that wild vertebrate reservoirs may host both pathogens and mutually cooperate to increase the supply of infective forms to sandflies and maintain the natural transmission cycle between vectors and vertebrates.
In conclusion, our findings corroborate the notion that the ER stress response embases the cellular processes involved in Leishmania and ICOV intracellular replication, and it enlights the fundamental molecular mechanisms that can govern the coinfection between phleboviruses and Leishmania.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/v14091948/s1, Figure S1: ER stress inhibitors do not affect Leishmania viability; Figure S2: XBP1 splicing is maintained for 24 h post-coinfection.; Figure S3: The upregulation of CHPT1 and CEPT1 is sustained for 24 h post-coinfection. Figure S4: The expression of ATF4 and NRF2 is sustained for 24 h post-coinfection.