Role of C3, C4b, CXCL10 and LCN2 in HIV associated white matter injury

1. Overview

Scientific Rationale and Importance: White matter abnormalities are one of the hallmark features of HIV-associated neurocognitive disorders (HAND), which affect over 50% of HIV-infected individuals despite effective combined antiretroviral therapy [1]. The oligodendrocyte injury is a hallmark in demyelination and white matter damage and such damage has been seen in HIV patients [2]. HIV-1 viral proteins, including the envelope glycoprotein 120 (gp120), trans-activator of transcription (Tat), and negative regulatory factor (Nef), have been implicated in HIV-1-associated oligodendrocyte injury [3-7]. In a previous microarray study conducted in our lab using whole-brain tissue from transgenic mice expressing HIV-1 gp120, we observed a marked upregulation of four inflammatory mediators: C3 (complement component 3), C4B (complement component 4B), CXCL10 (C-X-C motif chemokine ligand 10), and LCN2 (lipocalin-2), implicating them in the pathogenesis of HAND [8]. Upregulation of complement genes C3 and C4b has been widely studied in white matter injury during neurodegenerative disease [9, 10]. CXCL10 has been implicated in HIV related neuropathogenesis, acting as a chemoattractant that exacerbates neuroinflammation. Both CXCL10 and LCN2 were found to be significantly elevated in the brains of HIVgp120 transgenic mice [11-13]. Further, recent work from our lab using LCN2 knockout (KO) mice demonstrated that LCN2 plays a critical role in mediating neurotoxicity during HIV infection. Genetic ablation of LCN2 resulted in reduced microglial activation, preserved neuronal structure, and significantly improved behavioral outcomes in HIVgp120 transgenic mice, underscoring the translational relevance of this pathway.[14]. Thus, this proposal aims to elucidate the mechanistic contributions of C3, C4B, CXCL10, and LCN2 to HIV-induced white matter injury. Using a human iPSC-derived tri-culture system consisting of astrocytes, neurons, microglia, and oligodendrocytes, we will systematically evaluate the expression, regulation, and functional impact of these genes during HIV-1BaL infection.

Funding/Facilities/Ethics Approvals:

Kaul Laboratory: The proposed research will be conducted in Dr. Kaul’s well-equipped laboratory, located in approximately 1,500 sq. ft. on the third floor of the School of Medicine Research Building (SOMRB), at University of California, Riverside, providing a comprehensive environment for molecular biology, stem cell research, virology, and neurobiology. The lab includes wet bench space, adjacent tissue culture facilities suitable for handling human iPSC lines and HIV-1 virus, as well as access to a cold room, centrifuge room, autoclave, darkroom, and radiation room. Major equipment comprises Class II biosafety cabinets (BSL2 and BSL2+), CO₂ incubators, centrifuges, thermal cyclers, QuantStudio Real-Time PCR system, and deconvolution microscopy (Zeiss Axiovert 200M) with live cell imaging setup in a BSL2+ tissue culture room.

Shared Resources & Major Equipment:

These facilities are managed by the UCR School of Medicine and are accessible to all investigators. Most core instruments can be operated independently by trained laboratory personnel, or full-service analyses can be conducted by experienced core staff upon request:

Common equipment available to the laboratory includes: Departmental flow cytometry instrumentation (one FACScan and one FACSCalibur), one TomTec III cell harvester, one Pharmacia/Wallac Beta Plate reader, one Biorad qPCR instrument, one cryostat, and a Nanostring system.

SOM Instrumentation Core: The SOM operates three core facilities: 1) Histology Core for paraffin-embedded tissue processing; 2) Center for Intravital Imaging equipped with a Nikon A1R Multiphoton microscope housed in a dedicated rodent SPF vivarium; 3) Cell Sorter and Gene Expression Core featuring a MoFlo Astrios EQ Cell Sorter and 10x Genomics Chromium X system for high-throughput single cell and nucleus RNA-seq.

BSL-3 Facility: Additionally, the UCRSOM houses a 1,383 sq. ft. BSL-3 facility with three procedure rooms, equipped with biosafety cabinets, a pass-through autoclave, and specialized safety features. The facility is conveniently located adjacent to Dr. Kaul’s laboratory in the SOMRB to carry out HIV research. Biosafety approvals for HIV-Bal infection of CNS cell cultures have already been approved by UCR.

2. Introduction

Background and Literature: HIV-1 infection of the central nervous system (CNS) initiates a cascade of glial activation, inflammation, and myelin disruption, especially in subcortical white matter regions [7, 15]. These alterations persist despite viral suppression by combined antiretroviral therapy (cART). Complement proteins C3 produced by activated astrocytes can contribute to HIV mediated inflammation in brain and cognitive dysfunction [16]. However, C4B, a classical pathway component, has received limited attention in this context. Notably, our microarray analysis of HIVgp120 transgenic (HIVgp120tg) mouse brains revealed significant upregulation of C4B, alongside C3, CXCL10, and LCN2, suggesting potential involvement in the neuroinflammatory environment. The chemokine CXCL10 is elevated in HIV-infected glia and contributes to neuroinflammatory recruitment of toxic immune cells [17, 18]. LCN2, an acute-phase reactant secreted by astrocytes and microglia, exacerbates oxidative stress, blood-brain barrier dysfunction, and white matter damage in multiple models of CNS [19]. Our recent analysis of HIVgp120 transgenic mouse models and new findings from Favret et al. (2024) confirm that LCN2 knockout reduces gliosis, downregulates proinflammatory cytokines, preserves myelin, and improves cognition in neuroinflammatory diseases [14, 20]. Despite these findings, no study has yet combined measurement of C3, C4B, CXCL10, and LCN2 in human iPSC-derived mixed neuronal glial cell culture infected with HIV1-BaL, nor examined their collective neurotoxic impact on neurons.

Hypotheses:

H1: HIV-Bal infection of iPSC-derived astrocytes and microglia upregulates the expression and secretion of C3, C4B, CXCL10, and LCN2.

H2: The levels of C3, C4B, CXCL10, and LCN2 in glial supernatants correlate with the severity of neurotoxicity, such that high-secreting conditions induce greater neuronal damage, suggesting these factors are predictive biomarkers of HIV-associated glial-induced neurotoxicity.

H3: siRNA-mediated knockdown of C3, C4B, CXCL10, or LCN2 in glial cells reduces neuroinflammatory signaling and preserves neuronal viability in co-culture systems.

Impact Statement: White matter damage underlies cognitive decline in HIV-associated neurocognitive disorders (HAND), a debilitating consequence experienced by up to 50% of long-term HIV-positive individuals. Yet the cellular mediators of glia-driven myelin injury remain incompletely understood. By investigating how C3, C4B, CXCL10, and LCN2 coordinate neuroinflammation and white matter loss in a human-relevant iPSC-based model, this study will uncover mechanisms of white matter degeneration in HAND. The findings will provide novel therapeutic targets aimed at glial-inflammatory signaling, with potential relevance to other disorders characterized by chronic neuroinflammation and demyelination such as multiple sclerosis, traumatic brain injury, and stroke.

3. Methods

Materials and Procedures:

For Hypothesis 1 (H1): a) Cell Culture and Differentiation: Human iPSC-derived microglia, neurons and astrocytes will be obtained from commercial vendors like Fujifilm Cellular Dynamics International (CDI), BitBo, Neucyte or BrainXcells. Cells will be thawed and plated on Matrigel, PEI and PDL coated tissue culture plates (Corning) in maintenance medium as per manufacture instructions. Cells will be maintained at 37°C in a 5% CO₂ incubator.

b) Astrocytes and Microglia Co-culture System: Human iPSC-derived astrocyte and microglia will be co-cultured on poly-D-lysine (PDL) coated tissue culture plates, following the manufacturers’ instructions. After three days in co-culture, cells will then be infected with M-tropic HIV-1BaL at multiplicities of infection (MOI) of 0.1 for 4 hours. Following infection, cultures will be washed twice with PBS to remove unbound virus and replenished with fresh media. Uninfected control wells will undergo the same treatment, excluding virus exposure. Cell culture supernatants (condition media, CM) will be collected 0, 3, 6, 9, and 12 days post infection and assessed for HIV replication using a p24 ELISA (Abcam, HIV-1 p24 ELISA Kit) according to the manufacturer’s protocol. The cell culture supernatants will then be accessed for the increased secretion of C3, C4B, CXCL10, and LCN2 using commercially available ELISA kit from Abcam. Each experiment will have minimum of 3 replicates per experimental condition.

For Hypothesis 2 (H2): a) Neurotoxicity assay and analysis: The neurotoxicity assay will be performed as previous described by our lab [21]. Briefly, neuronal viability will be assessed using a co-culture model of iCell Neurons and iCell Astrocytes (Fujifilm CDI) seeded in black-walled, clear-bottom 96-well plates (Corning) pre-coated with 0.1% polyethyleneimine (PEI, Sigma-Aldrich) and Geltrex (Life Technologies) following manufacturer instructions. Cells will be maintained for 7 days with 50% medium changes every 2–3 days. Conditioned media (CM) from HIV-1Bal infected glial cultures will be transferred to these neuronal co-cultures for 24 h, followed by fixation in 4% PFA (25 min, 4 °C). Cells will be permeabilized with 0.2% Triton X-100, blocked in 10% heat-inactivated goat serum, and stained with Hoechst, anti-MAP2 (mouse, Sigma M4403), and anti-NeuN (rabbit, Millipore ABN78). Secondary antibodies include Alexa Fluor 488 (goat anti-rabbit, Invitrogen A11034) and Rhodamine Red (goat anti-mouse, Jackson 115-295-146). Imaging will be conducted using a Zeiss Axiovert 200M microscope with Slidebook software. Double-positive MAP2/NeuN neurons will be quantified, with vehicle-treated wells set as 100% survival. In parallel, ATP-based viability will be assessed using the CellTiter-Glo Luminescent Assay (Promega). Each condition will be tested in triplicate. CXCL10, C3, C4B, and LCN2 concentrations in CM (via ELISA) will be statistically correlated with neuronal survival percentages using Pearson correlation analysis.

b) Oligodendrocyte/White Matter Injury Assay: Oligodendrocyte progenitor cells (OPCs) will be purchased from Tempo Bioscience and cultured together with neurons and astrocytes according to the manufacturer’s instructions. Briefly, neurons and astrocytes will be seeded on PEI and Geltrex coated plates and maintained for 5 days with 50% medium changes every 2–3 days. On Day 5, OPCs will be introduced along with supplements (T3 and ascorbic acid) recommended by the manufacturer to promote OPCs maturation. The tri-culture will be maintained for 4 weeks to allow cells to reach the myelination phase, with half-medium changes every 2–3 days. After 4 weeks, conditioned media (CM) from HIV-1BaL infected glial cultures will be transferred to these tri-cultures for 24 h, followed by fixation in 4% PFA (25 min, 4 °C). Immunostaining will be performed for major myelin proteins, including myelin basic protein (MBP, Cell Signaling), proteolipid protein 1 (PLP1, Proteintech), and myelin oligodendrocyte glycoprotein (MOG, Santa Cruz). Quantitative image analysis will be used to assess the extent of myelination and myelin integrity following CM exposure from HIV-infected glial cultures. Cell viability will be evaluated in parallel using an ATP-based assay (CellTiter-Glo) as previously published [22].

For Hypothesis 3 (H3): a) siRNA Knockdown of C3, C4B, CXCL10 and LCN2: To evaluate the contribution of complement and inflammatory signaling to HIV-induced neurotoxicity, we will conduct siRNA knockdown of the following human genes: C3, C4B, CXCL10, and LCN2, in astrocytes and microglia. Pre-validated ON-TARGETplus SMARTpool siRNAs (Horizon Discovery / Dharmacon) will be used for each gene, along with a non-targeting siRNA pool as controls. siRNA transfection will be performed using Lipofectamine RNAiMAX or equivalent low-toxicity reagent according to manufacturer’s protocols. Astrocytes and microglia co-culture will be transfected independently for 48 hours prior to HIV infection. Following knockdown, total RNA will be extracted and analyzed by RT-qPCR to validate reduced expression (≥75%) using gene-specific primers for C3, C4B, CXCL10, and LCN2. Alternatively, protein lysates will be prepared and analyzed by Western blot using commercially available antibodies targeting all four markers.

b) HIV1-BaL Infection after siRNA Knockdown and neurotoxicity assay: Following confirmation of knockdown, siRNA-treated glial cells will be infected with HIV-1BaL at a MOI of 0.1, as described in Hypothesis 1. Three days post-infection, culture supernatants will be collected for HIV replication assessment using the HIV-1 p24 ELISA Kit (Abcam), following the manufacturer's protocol. Additional aliquots of supernatants will be stored at –80 °C for downstream applications, including neurotoxicity assays. Depending on the ELISA results from Hypothesis 1, the duration of HIV-1BaL infection may be extended, and additional supernatants collected at later time points post-infection. Simultaneously, cells will be lysed and stored for subsequent RNA or protein analysis, following protocols previously established by our lab. As outlined in Hypothesis 2, neurotoxicity assays and oligodendrocyte injury assay will be conducted using the stored supernatants from the siRNA knockdown experiments. These assays will determine whether silencing of C3, C4B, CXCL10, or LCN2 can reduce glia-induced neurotoxicity and preserve neuronal viability, thereby testing Hypothesis 3.

Ethics and Data Management: All procedures involving HIV-1Bal infections will follow institutional biosafety protocols. Results will be stored in secured lab servers. De-identified datasets and analysis scripts will be shared through public repositories after publication. This study will utilize commercially sourced human iPSC-derived neurons, astrocytes, microglia, and obtained from certified vendors that provide fully consented and de-identified cell lines. These vendors adhere to international ethical standards, including informed donor consent, IRB approval, and compliance with FDA/EMA regulations for the derivation and use of human pluripotent stem cells. This study does not involve any direct collection of human samples or identifiable human data, or any animal models. All experiments involving HIV-1BaL will be conducted strictly under Biosafety Level 2 (BSL-2+) conditions, in accordance with institutional biosafety protocols and CDC/NIH guidelines for working with HIV in cell culture. All personnel will be trained and certified in safe handling, decontamination, and waste disposal procedures. HIV work will be pre-approved by the institutional Biosafety Committee, and all materials will be autoclaved and treated before disposal.

Data Sharing Commitment:

In compliance with open science principles, data generated from this project will be made available upon publication or project completion via a public data repository (e.g., NCBI GEO or Figshare), following FAIR (Findable, Accessible, Interoperable, Reusable) data practices. Processed qPCR datasets, imaging data, and ELISA results will be archived, and metadata will include cell type, treatment condition, infection status, and time points. Detailed protocols will also be made publicly available to ensure reproducibility.

Timeline:

The project is designed to be completed within an 18 months’ timeline, as outlined below:

Timeline	Milestones
Month 1	Procurement of reagents and cells
Months 2-4	HIV-Bal Infection in microglia and astrocyte co-culture, ELISA for C3, C4B, CXCL10, and LCN2
Months 5-8	Neurotoxicity assay and optimization of siRNA transfection in astrocytes and microglia; knockdown validation via RT-qPCR or Western blot (WB)
Months 9-12	HIV-1BaL infection in siRNA-treated glia; collection of supernatants and Cells for RNA or WB
Months 13-15	Neurotoxicity and white matter injury assays using conditioned media
Months 15-18	Data analysis, figure preparation, and manuscript/report drafting

4. Pilot Data (Optional)

CXCL10 has been implicated in HIV neuropathogenicity and previous studies led by our lab using HIVgp120 transgenic (HIVgp120tg) mouse models have demonstrated significant upregulation of CXCL10 in various brain regions, such as the hippocampus and cortex, as a consequence of HIVgp120 expression, reflecting elevated neuroinflammatory signaling [11-14].

On the other hand, our lab has shown that LCN2 is a key mediator of HIVgp120-induced neurotoxicity. In mixed neuronal glial cultures, we have demonstrated that recombinant LCN2 causes a marked loss of MAP2⁺ neurons, and that this effect depends on microglial presence and p38 MAPK activation. Additionally, in vivo studies revealed that LCN2 knockout in HIVgp120 transgenic mice reduced gliosis, preserved neuronal integrity, and improved cognitive outcomes [14]. These findings provide strong in vivo rationale for analyzing CXCL10 and LCN2 in in vitro human iPSC derived brain cells. Although we have not previously focused on C3 and C4B, our microarray data from the HIVgp120tg mouse brain revealed marked upregulation of these complement genes, suggesting their potential involvement in HIV-related neuropathology. Thus, it is both scientifically relevant and novel to examine the roles of all four genes C3, C4B, CXCL10, and LCN2 in our current study. To demonstrate the feasibility of our proposed approach, we have conducted pilot studies using iPSC-derived glial and neuronal monocultures to establish baseline cell health and HIV infection protocols.

5. Budget

iPSC-derived astrocytes, neurons, microglia, OPCs media/plate supplies: $15,000
qPCR and ELISA kits (4 genes, time points): 5,000
Antibodies: $5,000
siRNA reagents: $4,000
Imaging reagents and cell stains: $1,000
Total estimated budget: $30,000

Budget Requested from Research Hub: 5000$

The project is supported by 25000 USD secured from other NIH grants, and the lab is already equipped with several of the necessary reagents, cells, instruments, to carry out the proposed research. We are requesting an additional 5000 USD from research ResearchHub to purchase ELISA kits.

Graphical Abstract:

6. References

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2. Liu, H., et al., Oligodendrocyte Injury and Pathogenesis of HIV-1-Associated Neurocognitive Disorders. Brain Sci, 2016. 6(3).

3. Hauser, K.F., et al., HIV-1 Tat and morphine have interactive effects on oligodendrocyte survival and morphology. Glia, 2009. 57(2): p. 194-206.

4. Bernardo, A., C. Agresti, and G. Levi, HIV-gp120 affects the functional activity of oligodendrocytes and their susceptibility to complement. J Neurosci Res, 1997. 50(6): p. 946-57.

5. Kimura-Kuroda, J., K. Nagashima, and K. Yasui, Inhibition of myelin formation by HIV-1 gp120 in rat cerebral cortex culture. Arch Virol, 1994. 137(1-2): p. 81-99.

6. Nukuzuma, S., et al., Exogenous human immunodeficiency virus-1 protein, Tat, enhances replication of JC virus efficiently in neuroblastoma cell lines. J Med Virol, 2012. 84(4): p. 555-61.

7. Radja, F., et al., Oligodendrocyte-specific expression of human immunodeficiency virus type 1 Nef in transgenic mice leads to vacuolar myelopathy and alters oligodendrocyte phenotype in vitro. J Virol, 2003. 77(21): p. 11745-53.

8. Maung, R., et al., CCR5 knockout prevents neuronal injury and behavioral impairment induced in a transgenic mouse model by a CXCR4-using HIV-1 glycoprotein 120. J Immunol, 2014. 193(4): p. 1895-910.

9. Festa, L.K., J.B. Grinspan, and K.L. Jordan-Sciutto, White matter injury across neurodegenerative disease. Trends Neurosci, 2024. 47(1): p. 47-57.

10. Zhang, L.Y., et al., Microglia exacerbate white matter injury via complement C3/C3aR pathway after hypoperfusion. Theranostics, 2020. 10(1): p. 74-90.

11. Singh, H., et al., Interferon-beta deficiency alters brain response to chronic HIV-1 envelope protein exposure in a transgenic model of NeuroHIV. Brain Behav Immun, 2024. 118: p. 1-21.

12. Singh, H., et al., A pivotal role for Interferon-alpha receptor-1 in neuronal injury induced by HIV-1. J Neuroinflammation, 2020. 17(1): p. 226.

13. Thaney, V.E., et al., IFNbeta Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury. Sci Rep, 2017. 7: p. 46514.

14. Ojeda-Juarez, D., et al., Lipocalin-2 mediates HIV-1 induced neuronal injury and behavioral deficits by overriding CCR5-dependent protection. Brain Behav Immun, 2020. 89: p. 184-199.

15. Kaul, M., G.A. Garden, and S.A. Lipton, Pathways to neuronal injury and apoptosis in HIV-associated dementia. Nature, 2001. 410(6831): p. 988-94.

16. Nitkiewicz, J., et al., HIV induces expression of complement component C3 in astrocytes by NF-kappaB-dependent activation of interleukin-6 synthesis. J Neuroinflammation, 2017. 14(1): p. 23.

17. Williams, R., et al., HIV-1 Tat co-operates with IFN-gamma and TNF-alpha to increase CXCL10 in human astrocytes. PLoS One, 2009. 4(5): p. e5709.

18. Mehla, R., et al., Programming of neurotoxic cofactor CXCL-10 in HIV-1-associated dementia: abrogation of CXCL-10-induced neuro-glial toxicity in vitro by PKC activator. J Neuroinflammation, 2012. 9: p. 239.

19. Zhang, J., et al., The role of lipocalin 2 in brain injury and recovery after ischemic and hemorrhagic stroke. Front Mol Neurosci, 2022. 15: p. 930526.

20. Favret, J., et al., Ablation of lipocalin-2 reduces neuroinflammation in a mouse model of Krabbe disease. Sci Rep, 2024. 14(1): p. 31822.

21. Koury, J., et al., EphB2-mediated ephrin-B reverse signaling on microglia drives an anti-viral, but inflammatory and neurotoxic response associated with HIV. Res Sq, 2025.

22. Sanchez, A.B., et al., Antiretrovirals, Methamphetamine, and HIV-1 Envelope Protein gp120 Compromise Neuronal Energy Homeostasis in Association with Various Degrees of Synaptic and Neuritic Damage. Antimicrob Agents Chemother, 2016. 60(1): p. 168-79.

Role of C3, C4b, CXCL10 and LCN2 in HIV associated white matter injury

Role of C3, C4b, CXCL10 and LCN2 in HIV associated white matter injury

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