There are over 100 medically relevant arboviruses recognized, and anyone who experiences an insect bite is at risk for exposure. Medically significant arboviruses are commonly found in the Togaviridae, Bunyaviridae, and Flaviviridae families. These families contain viruses that cause encephalitis or hemorrhagic fever, and vaccines or treatments are not available for most infections. Death rates are roughly 20,000–50,000 per year for any given arbovirus [1
]. While mortality rates are proportionately low to number of cases, the risk of permanent disability is as high as 50% [1
]. Rising temperatures and alterations in precipitation patterns have driven the emergence of these viruses into new regions [2
]. Often, when these viruses emerge, new symptoms and increased pathology occur [6
Infected individuals present with a spectrum of disease ranging from subclinical to death. The role of chronic diseases on intrinsic and innate immune defense is emerging as a significant player in a patient’s ability to respond to viral infections [12
]. Studies have shown that those with a chronic condition like Parkinson’s disease (PD) can have increased oxidative stress, which can in turn compromise the immune system [16
]. Other studies have shown that those with chronic neurological diseases specifically have altered inflammatory pathways, cytokine profiles, as well as autophagy dysregulation [18
]. Clinical studies have shown that viral infections can induce expression of pro-inflammatory cytokines that can impact mood and neurocognitive performance [20
]. Taken together, these studies show that the genetic background of the host can impact the severity and duration of sequelae.
Viral parkinsonism has been documented for a variety of human pathogens though there are few studies that evaluate the effects of viral infection on degenerative neurological diseases [1
]. Post-viral parkinsonism has been documented for several viruses, including Dengue virus [23
], West Nile virus [24
], Japanese encephalitis virus [25
], and St. Louis encephalitis virus [26
How viruses cause parkinsonism is not known. Animal models of neurological infections do not translate to nor mimic changes in the human cerebral cortex documented in postmortem reports and imaging studies. Evaluation of central spinal fluid in human and rodent studies indicate that an inappropriate neuroimmune response is responsible [27
]. This begs the question of how a virus might affect an individual with or without a predisposition to PD.
The use of human-induced pluripotent stem cells (hiPSC) in disease research is increasing not only because relevant data can be generated but because these cells can be differentiated systems that directly translate to a human model. Research on different hiPSCs and organoids has shown that products derived from patients with a disease state exhibit different morphology and gene expression compared with products derived from a normal patient [28
]. This research has deepened the understanding of disease states and highlighted potential issues for diseased persons [31
]. Studies have shown that neuronal hyperexcitability is found in organoids derived from patients with Alzheimer’s disease [35
]. Unfortunately, most studies utilizing PD cerebral brain organoids evaluate morphology but not systemic differences in innate immunity and neurotransmission [36
]. Most organoid modeling of PD is based on midbrain organoids that recapitulate PD pathologies of the dopaminergic networks, neurite disfunction, and abnormal localization of α-synuclein [37
Since significant differences in gene and protein expression exist between hiPSC lines derived from individuals with and without PD, we hypothesized that these differences could impact response to viral infection [38
]. This study aimed to classify those differences in response to infection with Chikungunya virus (CHIKV) so we might begin to understand how dysfunction in intrinsic and innate defenses could impact patient outcomes after infection with neurotropic arboviruses.
Research of viral encephalitis and other viral infections of the CNS are crippled by necessary ethical restraints. This field relies on autopsy findings, which are then typically applied to rodent models. Rodents do not present with symptoms of CNS pathologies unless they are genetically modified to be immune-deficient or have large quantities of virus administered via intracerebral injection or injection into other parts of the CNS. This has provided useful insights into the pathogenesis of these viruses but unfortunately has not translated to treatment or prevention of human disease.
While animals are valid and useful models, organoids could serve as a preliminary platform for screening that can better inform the design of animal studies and choice of genetic background. Ongoing advances with stem-cell research have provided a platform for producing specific cell types or organoids from human stem cells. Within the last few years, significant advances in human health have been made using stem cells and organoids [45
]. Human stem cells and organoids are emerging as a useful tool for virus research [50
]. Not only do they replicate cellular composition and expression of humans, but they are also less expensive, safer, and easier to use than animals. Using organoids as a model can also produce better data due to the ability to have more replicates and numbers per treatment.
When infected with CHIKV, non-PD and PD organoids produced similar amounts of virus for the same period, but after two weeks post-infection, PD organoids started to shrink. Immunofluorescence showed unique virus distribution patterns for non-PD and PD organoids. While non-PD organoids displayed uniform distribution of the CHIKV E2 protein, PD organoids exhibited local accumulation of CHIKV E2. This matches postmortem and necropsy data showing focal distribution of West Nile virus in brain tissue [52
]. Unfortunately, histological CNS data from CHIKV-infected humans is not available since the role of CHIKV in neurological disease is an emerging topic [6
]. This leads to the question of if the altered inflammatory response associated with PD contributes to the distribution of virus in the CNS and contributes to the establishment of chronic infection. In mice, CHIKV evades the CD8+
T-cell response to establish persistent infections [56
]. Gene expression data show that IL-12 and IL-18 activate the CD8+
T-cell response [56
]. It could be that the reduced expression of IL-12 and IL-18 in CHIKV-infected non-PD organoids is contributing to the infection patterns we observed. CD4 and CD8 cells would need to be incorporated into this model to delineate how the innate immune response is impacting the activation of T cells.
The distribution and density of astrocytes in non-infected organoids is comparable to other studies that document organoid morphology [57
]. GFAP was used to detect activated astrocytes. Histological studies have found that many mature astrocytes do not express significant GFAP unless activated [59
]. Thus, the lack of staining observed in non-infected non-PD organoids is likely indicative that astrocytes are in an inactivated state. Staining of PD organoids showed extensive distribution of astrocytes with a section of astrogliosis. Non-PD infected organoids had astrocytes distributed solely on the outer margins, which mimics other studies’ investigation astrocyte activation in organoids [57
]. Neurofilament is a component of mature neuronal cytoskeleton often found in high concentrations in axons. In growing or developing neurons, neurofilament may not be readily apparent since younger axons are much smaller than mature neurons [60
]. Imaging studies show neurofilament staining for all organoid types, which suggests that organoids possess mature neuron populations. Statistical analysis was not performed for these images due to the heterogeneous distribution of cell types (neuronal, glial, ependymal, etc.) between organoids.
SOX2 and Tuj1 were used to observe neuron proliferation in response to CHIKV. When compared to non-infected organoids, SOX2 fluorescence was similar in CHIKV-infected non-PD organoids but was reduced in CHIKV-infected PD organoids. SOX2 is a transcription factor that regulates pluripotency and neurogenesis and is integral to the growth and repair of neurons [61
]. The reduced expression of SOX2 in infected PD organoids could mean that there is dysfunction in neuron growth; however, detection of other assorted transcription factors would be necessary to understand the expression pattern.
∆∆Ct was used to analyze changes in gene expression. When infected organoids were compared to their respective non-infected controls, PD organoids showed a pattern of up-regulation, while non-PD organoids showed a pattern of down-regulation. While this is interesting in and of itself, this analysis did not provide much insight as to whether PD organoids were mounting an antiviral response that reflected the response of non-PD organoids. Previous work has documented that endogenous expression of most genes is different for PD and non-PD cells and patients [16
]. Thus, any changes, or lack thereof of PD organoids to viral insult may not reflect a typical antiviral response when compared to its non-infected counterpart. When ∆∆Ct analysis was performed comparing infected PD organoids with a non-PD non-infected control, it was observed that both non-PD and PD organoids had similar expression patterns for most markers in response to CHIKV infection. This indicates that PD organoids modify their expression in response to virus infection for most markers and mount a response like non-PD organoids.
This study showed an overall pattern of excitation of GABA, glycine, glutamate, and serotonin receptors in PD organoids when infected with CHIKV, while non-PD organoids exhibited a decreased pattern of expression for the same markers. While it is well documented that CHIKV can cause long-term or permanent depressive sequelae, there are no studies describing changes in neurotransmission. Two studies have reported the potential antiviral activity of serotonergic drugs on CHIKV replication though viral inhibition assays though impacts on serotonin neurotransmission are not described [62
]. GABRP, HTR3, SLC6A4, SLCA9, and SNPH were differentially expressed for both organoid types. HTR3 receptors are associated with neuro-gastrointestinal and psychiatric conditions that are controlled with 5-HT3 receptor antagonists [64
While the gene expression data shows that the response to CHIKV involves changes in neurotransmission and immune response, clinical trials have shown that dysregulation of the inflammatory response can remodel neurotransmission, leading the changes in mood and cognition [13
]. The reduced expression of ACHE has been linked to depression and cognitive deficits in human studies [67
]. Perhaps ACHE could be contributing to persistent depressive sequalae in CHIKV patients.
The data show that both PD organoids have increased expression of NFKB2, a transcription control protein that functions in the innate antiviral response [70
]. In PD, NFKB2 is activated with IL-17 when cultured with T-lymphocytes [71
]. Activation of NFKB2 results in the production of interferons, which play a significant role in the innate antiviral response [70
]. We also observed up-regulation of many proinflammatory cytokines in response to CHIKV infection in PD organoids when compared with non-PD organoids. CSF2 and CSF3 respond to infection by inducing inflammation and recruiting lymphocytes to the site of infection. Several viruses evade this immune response by blocking autophagy and thereby blocking monocyte differentiation and apoptosis [72
Oxidoreductases determine MHC class I surface exposure and influence the activation of inflammation cascades. When found on the plasma membrane, oxidoreductases signal intracellular stress status to the immune system [74
]. In particular, HMOX1 has antiviral activity with increased levels associated with clearance of infection [75
]. Our data show that PD organoids had increased expression of HMOX1, while non-PD organoids had decreased expression. When present, HMOX1 interacts with IL-10 (also down-regulated in infected non-PD organoids) as an anti-inflammatory mechanism of the innate immune response [78
]. Research has shown that Zika and Dengue viruses decrease host expression of HMOX1 as part of their antiviral response [75
]. The deficit of HMOX1 could be contributing to the persistence and pattern of CHIKV in the organoids.
Cytokines direct the innate immune response and play an important role in regulating the adaptive immune response. Specific cytokines can serve as biomarkers for viral infections [80
]. Our data support that PD organoids have increased expression of IL-5, IL-6, and IL-10, indicating that there could be an activation of a Th2 response. However, IL-12 was also significantly up-regulated in PD organoids, which would favor a cell-mediated inflammatory response to stress or infection as well as the activation of cytotoxic T lymphocytes. We observed significant down-regulation of IL-18 and IL-1B in non-PD organoids. These interleukins catalyze the production of several proinflammatory cytokines and recruit immune cells to the site of microbial infections. IL-12 promotes protective immunity to a variety of viruses, and IL-12 and IL-18 work together during the antiviral response [81
]. With IL-12 up-regulated and expression of IL-18 and IL-1 down-regulated, both PD and non-PD organoids could be having dysfunctional antiviral response. Of note, non-PD organoids had down regulation of IL-1 and IL-10 in response to CHIKV. This aligns with studies that have shown that reduced expression of IL-1 and IL-10 can exacerbate mental illness or psychotic episodes following infection with CHIKV [82
Chemokines are a subset of cytokines that are activated in response to tissue damage as well as foreign proteins and antigens. Overproduction of chemokines is associated with a variety of autoimmune diseases. Most chemokines we examined were expressed at greater levels in PD than non-PD organoids at 14 days post infection. This state of inflammation could potentially cause complications for responding to viral infections. CCL19 is a chemokine that binds to the CCR7 receptor and acts to recruit dendritic cells. CCL19 was up-regulated in PD and non-PD organoids. CCR7 was down-regulated in non-PD organoids but up-regulated in PD organoids. The expression profiles of PD organoids reflect expression profiles documented from cerebrospinal fluid from patients infected with Varicella–Zoster virus [85
]. Also, studies in CCR7-deficient mice reported increased death from West Nile virus infection via over-recruitment of leukocytes and inflammation [86
]. The reduced activity of CCR7 we observed could render organoids vulnerable to neuropathogens due to enhanced expression towards an inflammatory response.
CCL3 interacts with CCR4 and CCR5 during the acute inflammatory response and functions to recruit monocytes, which can have an impact on neuroimmunity [87
]. The increased expression of CCL3 and CCR5 in both PD and non-PD organoids also occurs during infection with respiratory pathogens and is associated with severe manifestations of disease [88
]. Animal studies support that expression of CCR5 is up-regulated in CNS infections with Japanese encephalitis virus and positively correlated with increased pathogenesis [89
]. Work has shown that increased levels of CCR5 contribute to demyelination and CNS disease [90
]. The increased expression of CCR5 in PD organoids indicates that they are in an inflammatory state or could be experiencing neuronal damage.
The complement system is a part of the innate immune response that can lyse cells, activate inflammation, target virus to phagocytic cells, and clear non-cytopathic viruses from the circulatory system. Here, we evaluated the expression of C3 as it functions in both classical and alternative complement activation pathways, and deficiency of C3 can make humans more susceptible to viral and bacterial infections [91
]. In our study, PD organoids had increased in C3 expression compared with non-PD organoids, suggesting a functional complement system. Non-PD organoids had down-regulation of C3, which has been reported in patients with hepatitis C infection [93
]. Functional expression of C3 is necessary to neutralize West Nile and other viruses which cause acute neurological infections and death [94
]. This poses an important question to be addressed in future research: could a reduction in C3 leave patients with CHIKV disease primed for neurological sequalae? Another future direction to build upon this study would be to conduct gain/loss of function studies on specific genes. Currently, disease modeling and the methodologies for conferring gain/loss of function in organoids of disease and non-diseased nature while also infected with a pathogen has not progressed to the point of reliability and reproducibility. This study presents key genes with altered expression and provides a foundation for these methodologies and studies to further develop.
The use of only two cell lines (one non-PD, one PD) is a limitation of this study due to the extensive genetic variation of PD. There are nearly 400 hiPSC cell lines derived from PD patients available for research [23
]. While typical neuronal studies utilize up to five cell lines per study (three diseased, two control), the appropriate numbers of cell lines to use for brain organoid research is still under debate [23
]. PD research utilizing organoids typically differentiate from one non-PD and one PD hiPSC line [47
]. In depth analysis of preliminary concepts requires substantial resources and time that is not justifiable for pilot studies, especially when generating organoids [23
]. Thus, preliminary data is often limited to two cell lines (control and diseased) [94
]. Regardless, the findings here need substantiation in organoids derived from additional cell lines. Of the two cell lines used in this study, one was obtained from the foreskin of a newborn, and one was obtained from a male donor aged 63 years. Studies in rodents have found age-related impact of viral infections, and observational studies in humans have reported specific pathogenesis in neonates [97
]. These studies above are reflective of whole organisms where DNA damage and telomere shortening are present in all somatic cells. Whole organisms also have functional immune systems that can contribute to pathogenesis. A benefit of hiPSC is that donor age does not affect the expression markers in differentiated cells since cellular rejuvenation occurs during the reprogramming of somatic cells into stem cells [99