In Vitro Analyses of the Multifocal Effects of Natural Alkaloids Berberine, Matrine, and Tabersonine against the O’nyong-nyong Arthritogenic Alphavirus Infection and Inflammation

O’nyong-nyong virus (ONNV) is a member of the reemerging arthritogenic alphaviruses that cause chronic debilitating polyarthralgia and/or polyarthritis via their tropism for the musculoskeletal system. Thus, the discovery of dual antiviral and anti-inflammatory drugs is a great challenge in this field. We investigated the effects of the common plant-derived alkaloids berberine (isoquinoline), matrine (quinolizidine), and tabersonine (indole) at a non-toxic concentration (10 μM) on a human fibroblast cell line (HS633T) infected by ONNV (MOI 1). Using qRT-PCR analyses, we measured the RNA levels of the gene coding for the viral proteins and for the host cell immune factors. These alkaloids demonstrated multifocal effects by the inhibition of viral replication, as well as the regulation of the type-I interferon antiviral signaling pathway and the inflammatory mediators and pathways. Berberine and tabersonine proved to be the more valuable compounds. The results supported the proposal that these common alkaloids may be useful scaffolds for drug discovery against arthritogenic alphavirus infection.

The antiviral potential of BER has been extended to CHIKV, SFV, and SINV [44,45]. Matrine (MAT) is a quinolizidine alkaloid isolated from various plants of the genus Sophora L. (Fabaceae family) with biological activities, including anti-inflammatory and antiviral effects, but no specific anti-alphaviral activity has yet been described [46]. Tabersonine (TAB) is an indole alkaloid isolated from the medicinal plant Catharanthus roseus (L.) G.Don (Apocynaceae family), which is endowed with potent anti-inflammatory activities [47].

Cytotoxicity
The viability of HS633T cells was assessed under infection by ONNV (MOI 1) or treatment by each of the three studied alkaloids (10 µM) in comparison with CHL (15 µM). The microscopy images (Figure 2A) showed morphological changes in the cells only when they were infected by ONNV. In the LDH and MTT assays, as shown in Figure 2B, no significant LDH release from the supernatants during infection or chemical treatment was detected, and the mitochondrial activity was not significantly affected. Thus, our further experiments were performed using the validated non-toxic concentrations. Berberine (BER) is an isoquinoline alkaloid derived from various plants, mostly from the genus Berberis (Berberidaceae family), with a wide range of biological activities [42,43]. The antiviral potential of BER has been extended to CHIKV, SFV, and SINV [44,45]. Matrine (MAT) is a quinolizidine alkaloid isolated from various plants of the genus Sophora L. (Fabaceae family) with biological activities, including anti-inflammatory and antiviral effects, but no specific anti-alphaviral activity has yet been described [46]. Tabersonine (TAB) is an indole alkaloid isolated from the medicinal plant Catharanthus roseus (L.) G.Don (Apocynaceae family), which is endowed with potent anti-inflammatory activities [47].

Cytotoxicity
The viability of HS633T cells was assessed under infection by ONNV (MOI 1) or treatment by each of the three studied alkaloids (10 µM) in comparison with CHL (15 µM). The microscopy images (Figure 2A) showed morphological changes in the cells only when they were infected by ONNV. In the LDH and MTT assays, as shown in Figure 2B, no significant LDH release from the supernatants during infection or chemical treatment was detected, and the mitochondrial activity was not significantly affected. Thus, our further experiments were performed using the validated non-toxic concentrations. structurally representative alkaloids; these are depicted in Figure 1. Their effects were compared to that of the 4-aminoquinoline chloroquine as a reference for antiviral and immunomodulatory effects [41].
Berberine (BER) is an isoquinoline alkaloid derived from various plants, mostly from the genus Berberis (Berberidaceae family), with a wide range of biological activities [42,43]. The antiviral potential of BER has been extended to CHIKV, SFV, and SINV [44,45]. Matrine (MAT) is a quinolizidine alkaloid isolated from various plants of the genus Sophora L. (Fabaceae family) with biological activities, including anti-inflammatory and antiviral effects, but no specific anti-alphaviral activity has yet been described [46]. Tabersonine (TAB) is an indole alkaloid isolated from the medicinal plant Catharanthus roseus (L.) G.Don (Apocynaceae family), which is endowed with potent anti-inflammatory activities [47].

Cytotoxicity
The viability of HS633T cells was assessed under infection by ONNV (MOI 1) or treatment by each of the three studied alkaloids (10 µM) in comparison with CHL (15 µM). The microscopy images (Figure 2A) showed morphological changes in the cells only when they were infected by ONNV. In the LDH and MTT assays, as shown in Figure 2B, no significant LDH release from the supernatants during infection or chemical treatment was detected, and the mitochondrial activity was not significantly affected. Thus, our further experiments were performed using the validated non-toxic concentrations.

Effects of Infection and Three Alkaloids on the Antiviral Type-I Interferon Signaling Pathway
The host cell intrinsic antiviral response was first studied from its initiation with the viral receptor MxRa8, the PRR RIG-I, the downstream signals of the IFNAR-dependent pathways (IFN-β), including the IRF3-dependent JAK/STAT pathway, and the resulting production of ISG-15.

Effects of Infection and Three Alkaloids on the Antiviral Type-I Interferon Signaling Pathway
The host cell intrinsic antiviral response was first studied from its initiation with the viral receptor MxRa8, the PRR RIG-I, the downstream signals of the IFNAR-dependent pathways (IFN-β), including the IRF3-dependent JAK/STAT pathway, and the resulting production of ISG-15.

Effects of Infection and Three Alkaloids on Inflammatory Mediators and Signaling Pathways
The anti-inflammatory potential of the studied alkaloids was first assessed on the key inflammatory mediators. When compared to the CTRL, as shown in Figure 5  was calculated using the Bonferroni multiple comparison test: *: p < 0.05, ****: p < 0.0001 when compared to non-infected cells (CTRL) and #: p < 0.05, ##: p < 0.01, ###: p < 0.001, ####: p < 0.0001 when compared to infected cells (ONNV).

Effects of Infection and Three Alkaloids on Inflammatory Signaling Pathways
The above results prompted us to investigate the effects of our alkaloids on the multiple alphaviral-related inflammatory signaling pathways, and the results are presented in Figure 6. . Effect on key inflammatory mediators. HS633T cells were infected with ONNV (MOI 1) and/or co-treated with chloroquine (CHL), berberine (BER), matrine (MAT), or tabersonine (TAB) for 24 h. RNA was collected and CCL2, CCL5, CXCL8, and TNF-α gene expression was determined by qRT-PCR analysis. Values are expressed as mean ± SEM of three independent experiments. pvalue was calculated using the Bonferroni multiple comparison test: *: p < 0.05, ****: p < 0.0001 when compared to non-infected cells (CTRL) and #: p < 0.05, ##: p < 0.01, ###: p < 0.001, ####: p < 0.0001 when compared to infected cells (ONNV).

Effects of Infection and Three Alkaloids on Inflammatory Signaling Pathways
The above results prompted us to investigate the effects of our alkaloids on the multiple alphaviral-related inflammatory signaling pathways, and the results are presented in Figure 6. RNA was collected and MAPK, NF-κB, STAT3, NLRP3, caspase-1, and COX-2 gene expression was determined by qRT-PCR analysis. Values are expressed as mean ± SEM of three independent experiments. p-value was calculated using the Bonferroni multiple comparison test: *: p < 0.05, ***: p < 0.001, ****: p < 0.0001 when compared to non-infected cells (CTRL) and #: p < 0.05, ##: p < 0.01, ###: p < 0.001, when compared to infected cells (ONNV).

Discussion
We surmised the anti-alphaviral potential of the alkaloids by their abilities to control the viral infection and to modulate the complex array of the innate host cell immune response [40]. In this work, we scrutinized and compared the multifocal effects of three pharmacologically relevant plant-derived alkaloids (berberine, matrine, and tabersonine) from three structural classes (isoquinoline, quinolizidine, and indole, respectively) ( Figure 1) against the potent reemergent arthritogenic alphavirus ONNV. Our study was performed using HS633T cells to reflect the tropism of arthritogenic alphaviruses for fibroblasts, as reported for CHIKV [14], with non-toxic concentrations of the alkaloid types (10 µM) and the continuously reevaluated antimalarial CHL (15 µM) as potent antivirals against reemerging viruses, at least in vitro (Figure 2).
Our results confirmed the infectivity of HS633T cells by ONNV (MOI 1), as established by the expression of critical viral E1, E2, capsid, and nsP2 genes ( Figure 3A). E1 and E2 glycoproteins are essential for virus entry [9,48]. All the non-structural proteins (nsP1-nsP4) are normally required for RNA synthesis [49], but the nsP2 protein is established as a virulence factor and inhibitor of the IFN-induced JAK/STAT pathway [48]. In addition, the essential interactions between the capsid protein and the E1 or E2 protein has also been underlined [50]. We showed that all the studied compounds efficiently downregulated E1 and E2 gene expression during ONNV infection, in spite of a distinct upregulation of capsid and nsP2 gene expression by MAT. Consistently, the viral progeny production was reduced when using BER or TAB ( Figure 3B). These results suggest the intracellular effect of the tested compounds, most probably by interference with viral replication.
As revealed by this study, ONNV infection triggers a robust host immune response from HS633T cells. The upregulation of MXRA8 grants effective viral entry [17]. The elevated expression of RIG-I (Figure 4) indicated that the infected cell was able to produce a robust antiviral response [51]. The gene expression of these host cell sensors for viruses was downregulated by BER and TAB, as it was for CHL, but a contrasting upregulation of MXRA8 was observed with MAT and at a level higher than that after ONNV infection. The activation of the type-I IFN antiviral response by HS633T cells was supported by the upregulation of the IFN-β and ISG15 gene expression during ONNV infection, and the studied compounds exerted distinct effects. The counterintuitive effects of MAT were reflected by the upregulation of the IRF3 and ISG15 gene expression, while drastically downregulating the IFN-β gene expression. Using CHL, BER, or TAB, there was no observable effect on the IRF3 gene expression, but there were significant effects on IFN-β and ISG15. BER increased the IFN-β gene expression but decreased the ISG15 gene expression, while TAB suppressed the expression of these two genes. The above observations suggest that the activation Pharmaceuticals 2023, 16, 1125 9 of 16 of the type-I IFN response occurred independently of IRF3. As also evidenced by our experiments, ONNV may be able to impair the STAT1-related antiviral signaling, as previously described for SINV in vitro [52] or CHIKV [14,53]. The STAT1 silencing during the alphavirus infection resulted from the host cell shutoff caused by the nsP2 protein, which affected the JAK-STAT signaling pathway [54]. None of the tested compounds was able to reinstate the STAT1 gene expression. The PI3K/AKT and AMPK-mediated autophagy has been established as a part of the defensive innate immune response for several Old World alphaviruses, including SFV, RRV, and CHIKV [24], but the pro-or antiviral functions of this intracellular process remain questionable [55,56]. We observed that this process was altered in HS633T cells by ONNV and reinstated only by CHL.
Acute to chronic inflammation is a result of alphavirus infection with mild to severe disorders, as supported for CHIKV [57][58][59][60]. We further observed a robust chemokine/cytokine response during ONNV infection resulting from the upregulation of the critical CCL2 (MCP-1), CCL5 (RANTES), and TNF-α inflammatory mediators ( Figure 5). CHL efficiently suppressed the gene expression of CCL2, CCL5, and TNF-α. The common effect of the other compounds was revealed by their remarkable downregulation of TNF-α. Their contrasting effects were observed on CCL2 gene expression, which was efficiently downregulated by BER and TAB but upregulated by MAT.
The analyses of the various inflammatory signaling pathways ( Figure 6) revealed the upregulation of NF-κB, NRLP3, caspase-1, and COX-2 gene expression during ONNV infection and a significant mRNA level for MAPK, but our data were inconclusive for STAT3. The involvement of the NF-κB pathway has been demonstrated in mature neurons during SINV infection in vitro [61]. NF-κB is involved in both the type-I IFN response [62] and the inflammatory response [63]. Interestingly, our three alkaloid types had no significant effect on NF-κB gene expression, while CHL induced its downregulation. The MAPK pathway is involved in lifecycle of the cell and the inflammatory responses [64], and it was reported that CHIKV induced MAPK expression in human osteosarcoma (HOS) cells [45]. Its downregulation during ONNV infection was consistent with a previous report for SINV in vitro in human neural progenitor (hNPC) cells [65], and its upregulation occurred only with TAB. The downregulation of STAT3 gene expression was also established in vitro for SINV in hNPC [65], but our data were inconclusive. The NLRP3/caspase-1 pathway favors the strong pro-inflammatory mediator release (IL-18 and IL-1β) and pyroptosis [66] and is related to the severity of the alphavirus infection. The upregulation of NRLP3 and caspase-1 by ONNV was consistent with previous reports on in vivo CHIKV infection [25] and in vitro and in vivo MAYV infection with high levels of IL-1β [26], and we established the remarkable regulatory effect of TAB. Finally, the upregulation of the COX-2 gene expression by ONNV in HS633T cells was consistent with the COX-2-mediated prostaglandin response induced by CHIKV infection in human synovial fibroblasts [22], and was significantly resolved by BER and TAB as well as by CHL.

Cell Infection and/or Treatment
The HS633T cells were cultivated in 6-or 96-well plates (100,000 cells/well and 10,000 cells/well, respectively) and maintained at 37 • C in a humid atmosphere with 5% CO 2 . The cells were allowed to grow until 80-90% confluence. The cells were then infected with ONNV to a multiplicity of infection 1 (MOI 1) and/or co-treated with CHL, BBR (Sigma-Aldrich, Darmstadt, Germany, 00900585), MAT (Sigma-Aldrich, Darmstadt, Germany, PHL89730), or TAB (Sigma-Aldrich, Darmstadt, Germany, SMB00452) for 24 h.

Statistical Analysis
The data were expressed as mean ± SEM of the three independent experiments. Statistical analysis was achieved using GraphPad Prism version 6.0 software. The p-value was calculated from variance analysis followed by a Bonferroni multiple comparison test.

Conclusions
Our overall results support the paradigm that the ONNV infection of fibroblasts drives a robust antiviral and inflammatory response via the essential type-I IFN pathway and canonical inflammatory signaling pathways (Figure 7). Our findings established the anti-alphaviral potential of the studied alkaloids by the control of viral expansion and the regulation of the host immune response. In-depth biological evaluation of potential antivirals is crucial for their profiling with regard to the immune response, as exemplified by the unexpected behavior of MAT. Finally, these alkaloids may be useful scaffolds for the discovery and development of novel anti-alphaviral drugs. regulation of the host immune response. In-depth biological evaluation of potential antivirals is crucial for their profiling with regard to the immune response, as exemplified by the unexpected behavior of MAT. Finally, these alkaloids may be useful scaffolds for the discovery and development of novel anti-alphaviral drugs. Figure 7. Effect of the studied alkaloids on immune responses (antiviral innate immunity: blue; inflammatory responses: pink). Alphaviruses (e.g., ONNV) use the receptor MXRA8 to bind to and infect fibroblast-like cells of connective tissues. Virus replicates inside the cells and single-stranded viral RNA can be detected by pattern recognition receptors (PRRs) such as RIG-I. Signaling pathways downstream from RIG-I coupled to MAVS activate transcription factors such as IRF3 and contribute to the increased production of type-I IFN (e.g., IFN-β). IFN-β binding to its receptor (IFNAR) activates JAK/STAT pathway and the transcription factors (STAT1/2) to promote the expression of antiviral interferon-stimulated genes (e.g., ISG15 and RNAse L). Viral infection also activates the expression of proinflammatory cytokines (e.g., TNF-α) and chemokines (e.g., CCL2) (notably via NF-κB and MAPK) and prostaglandin expression (via COX-2). The inflammasome contributes to the activation of caspase-1 and increased secretion of the canonical inflammatory cytokine IL1-β. The autophagy response can modulate viral replication and is under the control of two kinases (PI3K and AMPK). The inflammatory response contributes equally, on one hand, to the antiviral response but, on the other hand, if uncontrolled, to chronic injury of the joints.  Figure 7. Effect of the studied alkaloids on immune responses (antiviral innate immunity: blue; inflammatory responses: pink). Alphaviruses (e.g., ONNV) use the receptor MXRA8 to bind to and infect fibroblast-like cells of connective tissues. Virus replicates inside the cells and single-stranded viral RNA can be detected by pattern recognition receptors (PRRs) such as RIG-I. Signaling pathways downstream from RIG-I coupled to MAVS activate transcription factors such as IRF3 and contribute to the increased production of type-I IFN (e.g., IFN-β). IFN-β binding to its receptor (IFNAR) activates JAK/STAT pathway and the transcription factors (STAT1/2) to promote the expression of antiviral interferon-stimulated genes (e.g., ISG15 and RNAse L). Viral infection also activates the expression of proinflammatory cytokines (e.g., TNF-α) and chemokines (e.g., CCL2) (notably via NF-κB and MAPK) and prostaglandin expression (via COX-2). The inflammasome contributes to the activation of caspase-1 and increased secretion of the canonical inflammatory cytokine IL1-β. The autophagy response can modulate viral replication and is under the control of two kinases (PI3K and AMPK). The inflammatory response contributes equally, on one hand, to the antiviral response but, on the other hand, if uncontrolled, to chronic injury of the joints. Interestingly, alkaloids (in contrast to the unexpected effect of matrine) control viral infection. Alkaloids can also differentially modulate several steps of proinflammatory responses and open novel therapeutic avenues against alphavirus-induced acute and chronic pathologies. (Figure designed with Biorender).