Journal of Neuroinflammation

Yang et al. Journal of Neuroinflammation(2024) 21:145
https://doi.org/10.1186/s12974-024-03141-4

Open Access

RESEARCH

cFLIP in the molecular regulation of astrogliadriven neuroinflammation in experimental
glaucoma
Xiangjun Yang1, Qun Zeng1, Maide Gözde İnam1, Onur İnam1, Chyuan-Sheng Lin2 and Gülgün Tezel1*
Abstract
Background Recent experimental studies of neuroinflammation in glaucoma pointed to cFLIP as a molecular switch
for cell fate decisions, mainly regulating cell type-specific caspase-8 functions in cell death and inflammation. This
study aimed to determine the importance of cFLIP for regulating astroglia-driven neuroinflammation in experimental
glaucoma by analyzing the outcomes of astroglia-targeted transgenic deletion of cFLIP or cFLIPL.
Methods Glaucoma was modeled by anterior chamber microbead injections to induce ocular hypertension inmouse lines with or without conditional deletion of cFLIP or cFLIPL in astroglia. Morphological analysis of astroglia
responses assessed quantitative parameters in retinal whole mounts immunolabeled for GFAP and inflammatory
molecules or assayed for TUNEL. The molecular analysis included 36-plexed immunoassays of the retina and optic
nerve cytokines and chemokines, NanoString-based profiling of inflammation-related gene expression, and Western
blot analysis of selected proteins in freshly isolated samples of astroglia.
Results Immunoassays and immunolabeling of retina and optic nerve tissues presented reduced production ofvarious proinflammatory cytokines, including TNFα, in GFAP/cFLIP and GFAP/cFLIPL relative to controls at 12 weeks ofocular hypertension with no detectable alteration in TUNEL. Besides presenting a similar trend of the proinflammatory
versus anti-inflammatory molecules displayed by immunoassays, NanoString-based molecular profiling detected
downregulated NF-κB/RelA and upregulated RelB expression of astroglia in ocular hypertensive samples of GFAP/cFLIP
compared to ocular hypertensive controls. Analysis of protein expression also revealed decreased phospho-RelA andincreased phospho-RelB in parallel with an increase in caspase-8 cleavage products.
Conclusions A prominent response limiting neuroinflammation in ocular hypertensive eyes with cFLIP-deletion inastroglia values the role of cFLIP in the molecular regulation of glia-driven neuroinflammation during glaucomatous
neurodegeneration. The molecular responses accompanying the lessening of neurodegenerative inflammation
also seem to maintain astroglia survival despite increased caspase-8 cleavage with cFLIP deletion. A transcriptional
autoregulatory response, dampening RelA but boosting RelB for selective expression of NF-κB target genes, might
reinforce cell survival in cFLIP-deleted astroglia.
Keywords Astroglia, cFLIP, Cytokine, Glaucoma, Neuroinflammation*Correspondence:
Gülgün Tezel
gt2320@cumc.columbia.edu

1

Department of Ophthalmology, Vagelos College of Physicians andSurgeons, Columbia University, New York, NY 10032, USA
2
Department of Pathology & Cell Biology, Vagelos College of Physiciansand Surgeons, Columbia University, New York, NY, USA© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Yang et al. Journal of Neuroinflammation(2024) 21:145

Background
Complex neurodegeneration in glaucoma, a leading causeof blindness [1], encompasses a widespread inflammatory response of glial cells accompanied by progressive
loss of retinal ganglion cells (RGCs), optic nerve axons,
and their synaptic connections. Despite subtype-specific,
topographic, and temporal variations, glial inflammatory responses are evident throughout the visual pathway
from the retina to the brain. Although glial responses
may initially be beneficial and aid tissue clearance andhealing, a shift to chronic activation incites deleterious
neuroinflammation and promotes feedback loops, powering neuron loss. Besides increased production of proinflammatory neurotoxic molecules, withdrawal of the glial
mechanic, trophic, and bioenergetic support to RGCs
convoys glial dysfunction at this stage. Glia-driven stimulation of autoreactive T-cells, autoantibodies, and complement attack may also amplify the damaging stimuli
during glaucomatous neurodegeneration (reviewed [2]).
Therefore, elucidating the molecular players responsible
for regulating glial inflammatory responses remains anessential goal of glaucoma research to develop improved
treatments.
Recent studies of postmortem human tissues and animal models have highlighted a significant role for NF-κB
as a common transcriptional regulator of the multiple
inflammation pathways involved in glia-driven neuroinflammation and neurodegenerative outcomes, including TNFR and TLR signaling, and inflammasome [3–6].
Given the significant role of NF-κB in glia-driven inflammatory responses, following glaucoma studies have continued to explore related regulatory mechanisms further.
TNFα, a transcriptional target of NF-κB, is a major proinflammatory cytokine upregulated in the glaucomatous
glia [4, 7]. Upon death receptor activation by this specificligand, TNFR1 signaling, including caspase-8 as an initiator caspase, induces RGC apoptosis, oligodendrocyte
death, and axon degeneration [8–10].
Beyond promoting RGC death, caspase-8 regulates
glia-driven neuroinflammation by mediating glial cytokine production downstream of TNFR and TLR signaling. After death receptor or TLR activation, caspase-8
can raise NF-κB-regulated production of proinflammatory cytokines, including TNFα. Caspase-8 can also promote neuroinflammation by regulating inflammasome
involved in the proteolytic release of mature cytokines.
Besides its scaffold function for assembly of the signaling complex promoting NF-κB-dependent inflammation,
caspase-8 can activate various kinases and inflammatory mediators critical for inflammation signaling andinflammasome-driven processes [11–16]. Extensive
evidence has pointed to cFLIP (FLICE-like inhibitory
protein, also known as caspase-8 FADD-like apoptosis regulator, Cflar) that functions as a molecular switch

Page 2 of 19

between caspase-8-mediated inflammation, apoptosis, or
necroptosis pathways [17–19]. Recent experimental studies of glaucoma have also suggested that this caspase-8
homolog making a cytosolic complex with caspase-8
after TNFR or TLR binding regulates diverse processes
that control the cell type-specific roles of caspase-8 incell fate regulations [20]. After homo-oligomerization
and auto-processing of procaspase-8, its active form
proceeds to the execution of apoptosis via proteolytic
caspase cascade. While caspase-8 mediates RGC death,in astroglia, the caspase-8/cFLIP interaction prevents
apoptotic cleavage of caspase-8 but regulates the proinflammatory outcomes related to its dead effector domain(DED) or enzymatic activity. In light of this information, this study sought to determine whether deletion ofcFLIP can eliminate caspase-8-mediated inflammatory
responses of astroglia in experimental glaucoma. Analysis of neuroinflammatory outcomes in specific mouselines with conditional deletion of cFLIP (or the long isoform of cFLIP, cFLIPL) in astroglia valued cFLIP in themolecular regulation of glia-driven neuroinflammation
during glaucomatous neurodegeneration. In addition toa prominent immunomodulatory outcome of astroglial
cFLIP deletion, we detected a transcriptional autoregulatory response, including induction of RelB, reinforcing
cell survival.

Methods
Mice

This study included mouse lines with astroglia-targeting
conditional deletion of cFLIP. The first line targeting
glial fibrillary acidic protein (GFAP)-expressing astroglia (named GFAP/cFLIP) was generated by breeding thecFLIPflox [21] (B6.129-Cflartm1Ywh/J, Stock No: 022009;
The Jackson Laboratory, Bar Harbor, ME) [21] into
GFAP-cre/ERT2 (B6.Cg-Tg(GFAP-cre/ERT2)505Fmv/J,
Stock No. 012849; The Jackson Laboratory [22]), similar to previous studies [6, 20]. An additional line targeted the long isoform of cFLIP (cFLIPL) in astroglia.
The cFLIPLflox/flox mice were generated by Dr. ChyuanSheng Lin managing the Columbia University, Herbert
Irving Comprehensive Cancer Center Genetically Modified Mouse Model Shared Resource. The second line(named GFAP/cFLIPL) generated after cross-breeding ofcFLIPLflox/flox with GFAP-cre/ERT2 (The Jackson Laboratory) also allowed for the collection of isoform-specificinformation.
For conditional recombination, tamoxifen (dissolved incorn oil) was given to crossbreds by intraperitoneal injection (5 mg/40 g mouse) once a day for five consecutive
days starting a week before the experimental inductionof ocular hypertension. The age- and sex-matched controls (wild-type for the floxed allele with/without cre)

Yang et al. Journal of Neuroinflammation(2024) 21:145

received similar tamoxifen injections. Additional controls
included the littermates given the oil vehicle only.
PCR with primers detecting cre/ERT2 (5’-GCC AGTCTA GCC CAC TCC TT-3’, and 5’-TCC CTG AAC ATG
TCC ATC AG-3’), cFLIPflox (5’-CAT GAG CAC TGAGGG ACA CA-3’ and 5’-GCG GAG TTT GCT ACA
GGA AG-3’), or cFLIPLflox/flox (for the 1st loxp site, L83,
5’-tccaaaggttctaatgcctctt-3’, and 5’-gcagggactgttaactccataa-3’; and for the 2nd loxp site, FL146, 5’-catagggagaccctgtcatcta-3’, and 5’-atggcccatcactcacaaata-3’) alleles
verified the genotype. In addition, astroglial cre-recombinase immunolabeling and the analysis of cFLIP protein
expression confirmed recombination efficiency.
The mice were housed in a 12-hour light/dark cycle andreceived standard rodent chow and water ad libitum. All
animal experiments were conducted according to protocols approved by the Columbia University Institutional
Animal Care and Use Committee, and all procedures
complied with the Association for Research in Vision andOphthalmology (ARVO) statement’s tenets for using animals in ophthalmic and vision research.
Generation of cFLIP Lflox/flox mice

To generate the mice specifically lacking cFLIPL, the exon
6 of the cFLIP gene was flanked with two loxP sites (1stloxp: L83; 2nd loxp: FL146). Briefly, the Loxp-Neo-Loxp(LNL) cassette was inserted in the intron upstream of theexon 6 of the cFLIP gene on a Bacterial Artificial Chromosome (BAC clone ID: RP23-9D6) by BAC recombineering. The neo cassette was removed by cre recombinase toleave behind the 1st loxp site (L83). A Frt-Neo-Frt-Loxp(FNFL) cassette was inserted in the intron downstreamof exon 6 of the cFLIP gene. A gene targeting vector wasconstructed by retrieving the 2 kb left homology arm (5’to L83), the L83-exon6-FNFL cassette, and the 1.8 kbright homology arm (end of FNFL cassette to 3’) into thepUC57 vector carrying a Diphtheria Toxin Alpha (DTA)
chain. The linearized pUC57-Cflip-DTA with AscI waselectroporated into KV1 (129B6 hybrid) embryonic stem(ES) cells. Several targeted ES cell clones were identified. These targeted ES cells were injected into C57BL/6J
blastocysts to generate germline chimeric mice. Malechimeras were bred to homozygous Actin-beta (ACTB)(Flpe/Flpe) females (B6.Cg-Tg(ACTFLPe)9205Dym/J,
Stock No. 005703; The Jackson Laboratory) to transmit
the floxed cFLIP allele. A detailed floxed cFLIP sequenceis attached in SnapGene format. This sequence file canbe viewed by SnapGene viewer (https://www.snapgene.
com/snapgene-viewer/).
Modeling glaucoma in mouse

Mouse ocular hypertension was experimentally inducedby microbead injections into the anterior chamber[23], similar to previous studies [6, 20]. Briefly, 4 µl of aPage 3 of 19

mixture of 6 μm and 1 μm polystyrene microbeads, followed by 1 µl of 10 mg/ml viscoelastic sodium hyaluronate, was injected using a Hamilton syringe (Hamilton
Company, Reno, NV). To maintain ocular hypertension
for a period of up to 12 weeks, injections were repeatedat week 4. The fellow eye was similarly injected with anequivalent volume of physiologic saline. In this commonly utilized experimental model, neuroinflammation
develops as a consequence of induced ocular hypertension [6, 20].
Intraocular pressure was measured before and after
injections and then twice weekly using a TonoLab
rebound tonometer (TioLat, Helsinki, Finland) in isoflurane-anesthetized mice. Similar to detailed in previous studies [6, 20], despite steady intraocular pressurelevels below 12 mmHg in saline-injected fellow eyes,
microbead-injected eyes presented ocular hypertension(28.06 ± 4.12 mmHg) through the 12-week experimental
period. The mice with an intraocular pressure increase
above 40 mmHg were excluded from the study. The intraocular pressure-time integral was calculated for eachocular hypertensive eye as the mean intraocular pressure
over time corrected to the normotensive fellow eye tominimize any influence of intraocular pressure variability
among animals. Similar to previous studies [6, 20], ocular
hypertensive eyes included in this study had an intraocular pressure-time integral between 200 and 400 mm Hgdays, corresponding to up to 50% neuron loss.
Transgenic effects on glial morphological responses
were determined after 12 weeks of ocular hypertension
by analyzing the quantitative parameters in retinal whole
mounts immunolabeled for GFAP and inflammatory
molecules or labeled for the TUNEL assay.
The molecular analysis included 36-plexed immunoassays of retina cytokines and chemokines,
NanoString-based profiling of inflammation-related
gene expression in freshly isolated samples of astroglia,
and immunoblotting of astroglial proteins for cFLIP
expression, phospho-RelA/p65 or phospho-RelB expression, and caspase-8 cleavage. In addition, transgenic
effects on neurodegeneration were analyzed by RGC
counting.
Tissue immunolabeling

For morphological analyses of astroglia responses, theexpression pattern of the astroglial marker, GFAP, wasanalyzed as previously described [6, 20]. The wholemounted retinas fixed in 4% paraformaldehyde for 1 h
at room temperature were blocked in 1% BSA (SigmaAldrich) and 0.3% Triton X-100 (ThermoFisher Scientific;
Waltham, MA) for 1 h incubation. Besides a monoclonal antibody to GFAP (1:500; Abcam; catalog number:
ab68428 and ab10062), immunolabeling used a monoclonal antibody to TNFα (1:500; Abcam, catalog number:

Yang et al. Journal of Neuroinflammation(2024) 21:145

ab109322). Primary antibody incubation at 40C overnight was followed by another incubation using Alexa
Fluor dye (488, 555, or 647)-labeled secondary antibodies(1:1000; ThermoFisher Scientific). Slides were then coverslipped with 50 µl of Fluoroshield mounting medium
with 4′,6-diamidino-2-phenylindole, dihydrochloride(DAPI; Abcam, catalog number: ab104139), and images
were collected using the laser scanning confocal microscope (Red A1, Nikon Ti Eclipse; Nikon). Similar to previous studies, replacing the primary antibody with serum
was used as a negative control or an inappropriate secondary antibody for the species specificity. In addition towhole-mounted retinas, 6-µm thick histological sectionsof the paraffin-embedded optic nerve head tissues weresimilarly immunolabeled, as previously described [6, 20].
Confocal images of the GFAP-labeled retinal whole
mounts were analyzed for quantitative parameters
including the intensity (mean pixel intensity reflecting
the expression level) and percentage coverage of GFAP
labeling (the number of GFAP + pixels divided by the
total number of pixels, expressed as a percentage, reflecting the size and density of individual cells), as previously
described [6, 20] using ImageJ/FIJI (National Institutes of
Health, Bethesda, Maryland). To also determine the spatial distribution of GFAP immunolabeling, after setting
the scales to image scales and applying automatic thresholding, the region of interest (ROI) was manually traced
for the whole mount area. The center point of each image
was next defined as the center of the optic disc to draw acircular ROI with a 100-micrometer radius using X andY coordinates. 25 consecutive circles were then drawn
by increasing the radius from 100 micrometers to 2500
micrometers to obtain annular-shaped ROIs for assessing
the mean fluorescence intensity at a particular distancefrom the optic nerve head. Imaging-based quantitative
parameters of GFAP immunolabeling were analyzed
in a minimum of 14 mice per group, and the analysis ofastroglial TNFα immunolabeling utilized an additional
6 replicates per group. All analyses were conducted by aresearcher blinded to the experimental groups.
In addition, a Click-iT™ TUNEL imaging assay kit(ThermoFisher; catalog number: C10245) was utilized todetect apoptotic cells in retinal whole mounts, as previously used [20].
Multiplex protein quantitation

Retinal protein lysates were obtained by homogenization
in a lysis buffer (50 mM HEPES-KOH pH 8.0, 100 mM
KCl, 2 mM EDTA, 0.10% NP-40, 2 mM dithiothreitol,
10% glycerol, supplemented with protease and phosphatase inhibitors), and protein concentration was measured
with a colorimetric Bradford protein assay (BioRad, Hercules, CA), as previously described [6, 20]. Cytokine/
chemokine multi-analyte profiling was conducted with

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a ProcartaPlex 36-plex immunoassay using the Luminex
xMAP technology (ProcartaPlex TM mouse cytokine/
chemokine panel, Thermo Fisher Scientific). Analyses
included triplicated wells of experimental samples andcontrols, and the standards provided by the kit were used
to establish a standard curve for streamlined data analysis
with the ProcartaPlex Analysis App. Multiplex immunoassays were repeated three times using samples collectedfrom 3 mice per group in each set.
NanoString assay

For molecular profiling of the inflammation-related gene
expression, NanoString platforms (NanoString Technologies, Seattle, WA, USA) were used to count individual mRNA transcripts in isolated astroglia samples.
The astroglia isolation used the two-step immunomagnetic cell selection, as previously described [5, 6, 20]. In
the first step, a monoclonal CD11b (1:10; Abcam, catalog number: ab8878) was used to select microglia frompapain-dissociated retinas. The second step isolated
astroglia from the microglia/macrophage-depleted cellsuspension using a monoclonal antibody to GLAST1/
ACSA-2 (1:10; Miltenyl Biotech, catalog number 130099-138). RT-PCR and immunoblotting of selected
samples for specific markers verified this technique yielding ∼ 95% purity [5, 6, 20, 24]. Similar to previous studies, samples were pooled from multiple eyes matched for
cumulative intraocular pressure exposure (and neuron
counts within each group) [5, 6, 20].
RNA was extracted from freshly isolated samples
using a RNeasy Mini Kit (Qiagen, Germantown, MD),and Nanostring reactions were prepared according to
the manufacturer’s recommendation for the nCounter
Mouse Inflammation V2 Panel (NanoString Technologies). The panel consisted of 248 inflammation-related
genes and 6 internal reference genes (Cltc, Gapdh, Gusb,
Hprt, Pgk1, Tubb5), 8 negative and 6 positive controls
for normalization and the control of technical variability.
The RNA integrity was assessed in all samples by nucleic
acid fragmentation using a bioanalyzer (Agilent, Santa
Clara, CA), and the RNA quantity was assessed usinga Qubit fluorometer. Two hundred ng total RNA (RNAintegrity > 8.3; minimum concentration, 66–83 ng/µL)
per sample was submitted for hybridization and immobilizing cartridge processing. The gene expression data collected using the nCounter Digital Analyzer (NanoString
Technologies) were analyzed using the nSolver Analysis
Software 3.0 (NanoString Technologies). Heatmaps andvolcano plots were generated for group comparisons ingene expression profiling using the fold change in normalized molecular counts and z-scores. NanoString assay
used three samples collected from approximately 5 mice
per group in each set.

Yang et al. Journal of Neuroinflammation(2024) 21:145

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Immunoblotting

Results

Immunoblotting followed similar protocols to previous
studies [6, 20]. cFLIP expression was analyzed using a
monoclonal antibody (1:500; ThermoFisher Scientific,
catalog number: MA5-15739) To analyze caspase-8
cleavage, an antibody to procaspase-8 and cleaved subunits (1:500; ThermoFisher Scientific, catalog number:
13423-1-AP) was used. The primary antibodies also
included phosphorylation site-specific antibodies to
p65 [phospho-Ser536] (1:500; Abcam, catalog number:
ab76302) or RelB [phospho-Ser552] (1:500; ThermoFisher Scientific, catalog number: PA5-36837). In addition, a β-actin antibody (1:1000; ThermoFisher Scientific,
catalog number: MA5-15739, and Abcam, catalog number: ab179467) was used for loading and transferring
controls. After incubation with infrared dye-labeled secondary antibodies (1:10,000; Li-Cor, Lincoln, NE) for 1 h,proteins were visualized using Odyssey Infrared Imaging
system (Li-Cor), and the band intensity values were normalized to β-actin. Immunoblotting was repeated at least
three times with samples collected from approximately
40 mice per group.

Verification of mouse lines

RGC counting

RGCs were counted in retinal whole mounts immunolabeled for RNA-binding protein with multiple splicing(RBPMS, 1:200; GeneTex, Irvine, CA; catalog number:
GTX118619), as previously described [6, 20]. Laser scanning confocal microscopy (Red A1, Nikon Ti Eclipse;
Nikon, Melville, NY) and the NIS-elements AR5 software(Nikon) were used to acquire non-overlapping tile imagesof the whole-mounted retina for a depth of 0–30 μm asz stacks (with 5 μm step size) at a magnification of 10,
and images were collapsed into two-dimensional images
with maximum intensity projections for RGC counting.
RGC counting was conducted in a minimum of 16 mice
per group. All images were masked for the experimentalgroup of samples. Besides RBPMS labeling, RGC counting criterion included a minimal soma size of 10 μm toeliminate dying or phagocytized RGCs, as previously
described [6, 20].
Statistical analysis

Statistical analysis of the experimental data was carriedout in a masked fashion using SigmaPlot software (version 12.5; Systat Software, Inc., San Jose, CA). Groupdifferences were compared using the one-way analysis
of variance (ANOVA) followed by the pairwise multiple
comparison procedure of the Holm-Sidak method or
the Kruskal-Wallis one-way ANOVA on ranks followedby the Tukey test. The data are presented as mean ± SD
along with the p-values. A p-value of less than 0.05 wasconsidered significant.

This study tested whether conditional deletion of cFLIP
in astroglia can reduce neuroinflammation in experimental glaucoma. Mouse cFLIP codes two isoforms, cFLIPL
and cFLIPR [25, 26]. Figure 1 shows a diagram of thecFLIP gene in panel A. Besides targeted cFLIP deletion,in which exon 1 (used by both c-FLIPL and c-FLIPR) was
flanked [21], a new line was generated for the selective
deletion of cFLIPL by flanking the exon 6 of the cFLIP
gene with two loxP sites to determine isoform-specificdifferences. Panels B and C of Fig. 1 present the PCR
screening data, verifying the two loxP sites, L83 andFL146, by agarose gel electrophoresis of PCR products
in cFLIPL mutants. PCR genotyping also verified GFAPCre/ERT2 (500 bp) and mutant cFLIP (200 bp). In addition, astroglia-targeting cre/lox-based deletion efficiency
was tested by cre recombinase immunolabeling in GFAPexpressing astroglia in retinal whole mounts (Fig. 1D).
Figure 1 also presents immunoblots of the isolated
astroglia samples verifying the change in cFLIP protein
expression. Although astroglia from controls exhibitedcFLIP expression, including both cFLIPL and cFLIPR isoforms, no prominent cFLIP or cFLIPL immunoreactivity
was detectable in transgenic samples. As presented inpanel E of Fig. 1, immunoblots of retinal astroglia proteins detected no band corresponding to long (∼ 60 kDa)or short (∼ 30 kDa) isoforms of cFLIP in GFAP/cFLIP.
However, retinal astroglia proteins isolated from GFAP/
cFLIPL presented ∼ 30 kDa immunoreactivity corresponding to cFLIPR. These data collectively support theeffectiveness of targeted deletions in astroglia.
Transgenic effects on cellular apoptosis in cFLIP-deleted
astroglia

Since cFLIP regulates caspase-8-mediated cell death andinflammation pathways, Western blotting first focusedon apoptotic caspase-8 activity in the cFLIP-deleted
astroglia. Western blot analysis testing caspase-8 expression in the isolated astroglia proteins detected somecleavage products in ocular hypertensive samples besides
the full-length 55 kDa inactive proenzyme. In addition top55 procaspase-8 and its p43 cleavage product in control
samples, immunoblots detected p18 in cFLIP-deleted
ocular hypertensive samples. Figure 2 presents these
immunoblots and the related quantitative data (panel
E). Despite increased caspase-8 cleavage, no transgenic
effect was detectable on cellular apoptosis in astroglia.
As shown in Fig. 2A, there was no change in TUNEL ofastroglia between ocular hypertensive GFAP/cFLIP or
GFAP/cFLIPL and ocular hypertensive controls. Some
TUNEL + cells were detectable in the underlying layerof RGCs; however, this labeling was not associated with
GFAP-labeled astroglia (Fig. 2B). Instead, additional

Yang et al. Journal of Neuroinflammation(2024) 21:145

Page 6 of 19

Fig. 1 Generation of GFAP/cFLIPL mice. (A) Diagram of the cFLIP gene. (B) PCR screening. After breeding of the male chimeras and the ACTB(Flpe/Flpe)females, screening of 8 pups for L83 and FL146 by agarose gel electrophoresis of PCR products showed loxp heterozygous male (#6 and #8). After pairing
the heterozygous males with C57BL/6J females, screening of 8 pups from crossbreeding of the heterozygous mice showed loxp homozygous (#5). (C)Continued genotyping verified L83 and FL146 in cFLIPL mutants. Additional PCRs show GFAP-Cre/ERT2 (500 bp) and mutant cFLIP (200 bp). (D) Cre recombinase expression (red) was also analyzed in retinal GFAP-expressing astroglia (green) by specific immunolabeling of retinal whole mounts. White boxed
areas are shown in higher magnification, and the bottom panel shows the merged images of GFAP (green) and cre recombinase (red) immunolabeling(scale bar, 100 μm). (E) Immunoblots of retinal astroglia proteins detected no band corresponding to long (∼ 60 kDa) or short (∼ 30 kDa) isoforms of cFLIP
in GFAP/cFLIP. However, the short isoform was detectable in GFAP/cFLIPL. Also note increased band intensities in experimental glaucoma (G) samples
compared to non-glaucomatous controls (C). Graphs present data (mean ± SD) from quantitative analysis of the band intensities normalized to β-actin

analysis demonstrated colocalization of TUNEL with
RBPMS, a cell marker for RGCs (Fig. 2C) known toundergo apoptosis in ocular hypertensive eyes. As a positive control, DNase I treatment of the whole-mounted
retina resulted in TUNEL of retinal cells, including
GFAP + astroglia (Fig. 2D). In support of the maintained
survival of astroglia after targeted deletion of cFLIP orcFLIPL, further analysis in GFAP/cFLIP or GFAP/cFLIPL
mice detailed below also demonstrated protected cellstructure and morphological features accompanied bycontinued gene and protein expression in these cells.
Transgenic effects of the ocular hypertension-induced
inflammatory responses of astroglia with cFLIP, or cFLIPL,
deletion

Next, to determine transgenic effects on astrogliadriven neuroinflammation, alterations in morphological and molecular responses of astroglia were analyzed
in GFAP/cFLIP, GFAP/cFLIPL, and control mice with orwithout experimentally induced glaucoma. When the
GFAP immunolabeling pattern of astroglia was studied
in whole-mounted retinas, reactive astroglia in ocular
hypertensive samples exhibited increased intensity andpercentage coverage of GFAP labeling as expected. Compared to ocular hypertensive cFLIPf/f or cFLIPLf/f, ocular
hypertensive GFAP/cFLIP or GFAP/cFLIPL presented
faint alterations in these quantitative parameters. The
intensity of GFAP immunolabeling, reflecting the level of

GFAP expression, presented only a subtle drop in ocular
hypertensive GFAP/cFLIP and GFAP/cFLIPL relative toocular hypertensive controls. Similarly, ocular hypertension-induced increase in the percentage GFAP coverage,
reflecting the size and density of individual cells, slightly
decreased with astroglial cFLIP or cFLIPL deletion. However, neither these morphological data nor the spatial
distribution of the GFAP immunolabeling detected nosignificant difference between transgenic and control
groups (p > 0.05). Figure 3 exemplifies GFAP immmunolabeling in control and transgenic groups (panel A) andpresents the quantitative image analysis data (panels B
and C).
The study also included cytokine and chemokine profiles in the retina and optic nerve samples using multiplex immunoassays. Luminex-based analysis showeddecreased titers of multiple cytokines and chemokines inocular hypertensive GFAP/cFLIP and GFAP/cFLIPL micecompared to ocular hypertensive controls at 12 weeks
of ocular hypertension. The proinflammatory cytokines,
including IL1, IL2, IFNγ, and TNFα, presented significantly (p < 0.001) lower titers, while the titers of the antiinflammatory cytokines, including IL4 and IL10, weresignificantly (p < 0.001) increased in GFAP/cFLIP and
GFAP/cFLIPL. Figure 4 shows the related data.
To support the immunoassay data, the following analyses included tissue immunolabeling. TNFα immunolabeling was analyzed in retinal whole mounts and optic nerve

Yang et al. Journal of Neuroinflammation(2024) 21:145

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Fig. 2 Analysis of astroglial apoptosis. A. No alteration was detectable in TUNEL (red) of GFAP + astroglia (green) in retinal whole mounts from ocular
hypertensive GFAP/cFLIP or GFAP/cFLIPL compared to ocular hypertensive controls, cFLIPf/f or cFLIPLf/f (scale bar, 100 μm). When repeated in a minimum of
6 samples per group, TUNEL did not detect astroglia apoptosis. Blue shows DAPI nuclear stain. B. Some TUNEL + cells, not associated with GFAP-labeledastroglia, were detectable in some regions of the underlying layer of retinal ganglion cells (white arrows). C. Although GFAP-labeled astroglia were negative for TUNEL, additional analysis demonstrated TUNEL in some RBPMS (green)-labeled retinal ganglion cells (white short arrows). D. As a positive control,
DNase I treatment of the whole-mounted retina resulted in TUNEL of retinal cells, including GFAP + astroglia (white thin arrows). E. Despite no alterationin astroglia TUNEL with cFLIP deletion, Western blot analysis detected an increase in caspase-8 cleavage products in experimental glaucoma (G) samples
compared to non-glaucomatous controls (C). Besides the full-length 55 kDa inactive proenzyme, immunoblot analysis of caspase-8 expression detected
p43 and p18 subunits in astroglia samples isolated from ocular hypertensive GFAP/cFLIP. Immunoblotting was repeated at least three times with samples
collected from approximately 40 mice per group. The graph presents the fold change data (mean ± SD) from quantitative analysis of the band intensities
normalized to β-actin

sections to assess the proinflammatory phenotype ofastroglia. As shown in Fig. 5, a prominent decrease was
detectable in the TNFα labeling of GFAP + astroglia in
ocular hypertensive GFAP/cFLIP and GFAP/cFLIPL micerelative to ocular hypertensive controls. Quantitative
analysis indicated an approximately three-fold decreasein TNFα immunolabeling of the retina (Fig. 5A and B)
and optic nerve (Fig. 5C) in GFAP/cFLIP and GFAP/
cFLIPL. Figure 5 also presents the quantitative data fromimage analysis of TNFα immunolabeling in the retina andoptic nerve tissues, indicating an up to four-fold decrease
with cFLIP or cFLIPL deletion in astroglia (p < 0.001).
Thus, deletion of cFLIP, or cFLIPL, limited proinflammatory responses of astroglia.

Additional molecular profiling in cFLIP-deleted astroglia

NanoString-based profiling of the inflammation-related
genes explored the molecular responses of astroglia further. Transcripts from multiple genes were significantly
upregulated or downregulated in astroglia samples isolated from GFAP/cFLIP mice compared to controls.
Heatmaps and volcano plots of the molecular data arepresented in Fig. 6. Compared to ocular hypertensivecFLIPf/f controls, the retinal astroglia isolated from ocular hypertensive GFAP/cFLIP presented a significant
increase or decrease in 46 out of 248 genes studied. The
molecular alterations exhibited a trend parallel to that
detected by immunoassays and tissue immunolabeling. Significantly increased molecules in cFLIP-deleted
astroglia included an anti-inflammatory cytokine, Il10

Yang et al. Journal of Neuroinflammation(2024) 21:145

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Fig. 3 Morphological analysis of the astroglia response. (A) Retinal whole mounts immunolabeled for GFAP showed subtle alterations in the quantitative
morphological parameters of astroglial reactivity in ocular hypertensive GFAP/cFLIP and GFAP/cFLIPL compared to ocular hypertensive controls (cFLIPf/fand cFLIPLf/f). (B) Graphs show the intensity and the percentage coverage of GFAP immunolabeling in different groups. The data (mean ± SD) represent aminimum of 14 mice per group. Both GFAP intensity and coverage were not significantly different in ocular hypertensive GFAP/cFLIP or GFAP/cFLIPL than inocular hypertensive controls (one-way ANOVA; p > 0.05). (C) The studied quantitative parameters of GFAP immunolabeling did not also present significant
spatial alterations (one-way ANOVA; p > 0.05)(p = 0.01), while several proinflammatory cytokines,
including Il12 (p = 0.03), Il15 (p = 0.01), and Il22 (p = 0.04)
were decreased. Other molecules decreased in cFLIPdeleted astroglia included a regulator molecule of TNFαsignaling, Tnfaip3 (p = 0.003), and nos2 (p = 0.01). Several
complement components were among the moleculeswith increased expression in cFLIP-deleted astroglia.
An exciting outcome of the NanoString-based geneprofiling in ocular hypertensive samples included NF-κB
components, as RelA (p65) decreased (p = 0.002) and
RelB (p = 0.02) showed increased RNA counts in GFAP/
cFLIP mice compared to controls. To further evaluate
these molecular responses, Western blot analysis evaluated protein expression in isolated astroglia samples
from GFAP/cFLIP, GFAP/cFLIPL, and controls. Whenphosphorylation site-specific antibodies were used toanalyze phospho-RelA (p65) and phospho-RelB expression, retinal astroglia exhibited decreased expressionof phospho-RelA and increased expression of RelB in
ocular hypertensive samples of GFAP/cFLIP comparedto ocular hypertensive controls. Figure 7 exemplifies
related immunoblots and presents the statistical comparison of the protein expression in transgenic and control
samples. Besides alterations in proinflammatory versus
anti-inflammatory molecules, these findings support atranscriptional autoregulatory response, reducing RelA
but enhancing RelB in cFLIP-deleted astroglia.
Transgenic effects on neuron survival in experimental
glaucomaTo determine transgenic effects on the status of ocular hypertension-induced neurodegeneration, RBPMSlabeled RGCs were counted in whole-mounted retinas.
Figure 8 exemplifies RBPMS immunolabeling of retinal
whole mounts in control and transgenic groups (panelA) and presents the comparative RGC loss data (panel B).
RGC counting detected a greater number of remaining
RGCs in ocular hypertensive GFAP/cFLIP (42,231 ± 4,079;∼23% neuron loss versus 34,562 ± 3,503; ∼36% neuron
loss; p < 0.001) and GFAP/cFLIPL (41,229 ± 3,986; ∼24%
neuron loss versus 32,671 ± 3,543; ∼39% neuron loss;
p < 0.001) mice than ocular hypertensive controls. As
opposed to the RGC loss in controls, these values correspond to approximately 36% and 39% protection in
GFAP/cFLIP and GFAP/cFLIPL, respectively.

Discussion
Although therapeutic immunomodulation is a logical strategy to avoid inflammatory neurotoxicity in several neurodegenerative diseases, including glaucoma,

Yang et al. Journal of Neuroinflammation(2024) 21:145

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Fig. 4 Cytokine/chemokine profiling. Multiplex immunoassays of retina and optic nerve proteins detected altered titers of multiple cytokines and chemokines in ocular hypertensive GFAP/cFLIP and GFAP/cFLIPL mice than ocular hypertensive controls. Graphs present the fold change in ocular hypertension-induced alterations in 36 molecules studied in GFAP/cFLIP, GFAP/cFLIPL, and controls. The data (mean ± SD) represent a minimum of 3 samples
collected from 3 mice per group in each set. Green and red asterisks show significantly decreased or increased molecules, respectively, and accompanying tables list these molecules along with the p-values of group comparisons using one-way ANOVA

molecular regulation of neuroinflammation has yet tobe fully elucidated to identify effective treatment targets. Based on recent research findings from the studies of animal models and the postmortem tissues ofglaucomatous human donors, upregulated moleculesin the glaucomatous glia include several inflammatory
mediators linked to TNFR signaling, TLR signaling, andinflammasome activation [2, 27]. Among the glia-produced proinflammatory cytokines that function as effectors of neuroinflammation, TNFα comes forward as aprominent contributor to the proinflammatory-to-antiinflammatory cytokine imbalance in human glaucoma
and experimental models [4, 7]. It has also become evident that besides the direct neurotoxicity of TNFα to
RGCs through dead receptor signaling, downstream
NF-κB activation and transcription of immune mediators

amplify the vicious cycle of neurodegenerative inflammation driven by glia. A more recent study of experimental
glaucoma in mice with astroglia-targeting cre/lox-based
conditional inhibition of NF-κB activation supported a
critical role for this transcriptional activator in neuroinflammatory outcomes [6]. The following glaucoma studies have also built a scientific basis for a regulatory role ofcFLIP, a gene target of NF-κB, in glia-driven neuroinflammation. Upon activation of immune receptors, the interaction of DED-containing proteins, caspase-8 and cFLIP,
appears to constitute a signaling checkpoint for cell fatedecisions. [20] The current study focused on investigating the importance of cFLIP for astroglia-driven neuroinflammation in experimental glaucoma using conditionalcFLIP knockouts. Besides cFLIP, this study analyzed theoutcomes of the selective deletion of cFLIPL in astroglia

Yang et al. Journal of Neuroinflammation(2024) 21:145

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Fig. 5 Tissue immunolabeling of TNFα. (A) GFAP (green) and TNFα (red) immunolabeling of retinal whole mounts demonstrated a prominent decreasein ocular hypertensive GFAP/cFLIP compared to ocular hypertensive controls ocular hypertensive controls (cFLIPf/f). (B) GFAP (green) and TNFα (red) immunolabeling of retinal whole mounts was similarly decreased in ocular hypertensive GFAP/cFLIPL compared to ocular hypertensive controls. (C) Optic
nerve sections similarly exhibited decreased TNFα immunolabeling in ocular hypertensive GFAP/cFLIP. The merged images of the white boxed areas areshown in higher magnification. Images are representative of a minimum of 6 samples per group (scale bar, 100 μm). Graphs present data (mean ± SD)
from quantitative analysis of TNFα immunolabeling in GFAP + astroglia in transgenic and control samples. The intensity of astroglial TNFα immunolabelingwas significantly lower in ocular hypertensive GFAP/cFLIP or GFAP/cFLIPL than in ocular hypertensive controls (one-way ANOVA; p < 0.001)

since the balance between different isoforms, cFLIPL andcFLIPR, appears to impact the regulatory activities [28,29]. The findings of this study demonstrated a decreased
inflammatory response in experimental glaucoma with
cFLIP, or cFLIPL, deletion in astroglia. This immunomodulatory outcome was accompanied by regulatory molecular responses maintaining astroglia survival. Lesseningof neuroinflammation by targeted deletion of astroglial
cFLIP, or cFLIPL, provided RGC protection in ocular
hypertensive eyes, supporting the potential for immunomodulation in glaucoma treatment.
Caspase-8/cFLIP interaction in astroglia

This study, stimulated by recent experimental findings implying caspase-8/cFLIP interaction in cell fatedecisions in experimental glaucoma [20], assessed anessential role of cFLIP in the molecular regulation of gliadriven neuroinflammation. In the extrinsic pathway of
RGC apoptosis in glaucoma, which can be triggered by
the ligation of dead receptors, such as TNFR1 [7] or Fas[30], caspase-8 plays an initiator role. After the engagement of dead receptors, the initial membrane-boundcomplex rapidly signals for receptor-interacting protein
kinase 1 (RIPK1)-mediated activation of NF-κB that
promotes cell survival and proinflammatory signaling.
In contrast, a cytoplasmic complex formed in a second

step promotes apoptosis. Recruitment of procaspase-8
to the death-inducing signaling complex leads to homooligomerization and initiates an autocatalytic multistep
cleavage process, producing the mature enzyme. The
autoactivated caspase-8 cleaves effector caspases in thedownstream apoptosis cascade [31, 32]. However, procaspase-8 zymogen and its partially cleaved form canalso exert pro-survival and proinflammatory activities
through RIPK1, while preventing the necroptotic RIPK
activity [11, 12, 33–35]. Besides TNFRs, glial TLRs [3,5, 36] and inflammasome [4, 5, 37, 38], which are linked
to sensing tissue stress/damage, can also promote neuroinflammation in glaucoma. Like TNFR signaling, TLR
signaling [12, 14, 39–42] and inflammasome activation[15, 16, 43–46] involve caspase-8 in increased expression, proteolytic activation, and secretion of proinflammatory cytokines. By functioning as a scaffold for RIPK1
recruitment to the signaling complex, procaspase-8
plays an essential protease-independent catalytic rolefor NF-κB activation and the proinflammatory output[47]. Caspase-8 can also activate various kinases criticalfor inflammation signaling, such as including RIPK, NIK,
and IκKs, and other inflammatory mediators involved ininflammasome-driven processes [11–16].
After death receptor engagement, the rapidly activated
NF-κB by complex I also enables anti-apoptotic NF-κB

Yang et al. Journal of Neuroinflammation(2024) 21:145

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Fig. 6 NanoString-based molecular analysis of the astroglia response. NanoString profiling of 248 inflammation-related genes in isolated astroglia samples detected multiple genes significantly upregulated or downregulated in ocular hypertensive GFAP/cFLIP mice (1–3) compared to ocular hypertensive
controls (C1-3). The heatmap reflects z-scores for molecules with significantly changed expression. The volcano plot presents the fold change data. The
dotted line shows significance set at p < 0.05; the red data points above the reference line represent increased, and the green data points represent decreased expression. NanoString assay used a minimum of three samples collected from approximately 5 mice per group in each set

targets, including cFLIP, which interfere with the caspase-8 in complex II. As a DED-containing protease-deficient caspase-8 homolog, cFLIP regulates the dynamics
of the caspase-8 recruitment to complex II and inhibits apoptosis signaling by preventing its autocleavagemediated activation. While procaspase-8 presents a highaffinity to bind this signaling complex, the recruitmentof cFLIP is via heterodimerization with procaspase-8.
Due to the high efficiency of caspase-8 binding to complex II, cFLIP mainly prevents the enzymatic activity of
caspase-8 rather than its binding to this complex [48,49]. Evidently, both c-FLIPL and c-FLIPR block caspase-8
activation at different levels of procaspase-8 processing at
the complex II [50]. Several models have been reported
for the binding of cFLIP isoforms to this complex.
According to a recent model, their cooperative hierarchical binding to procaspase-8, depending on the unboundc-FLIPL to cFLIPS(R) ratio, is thought to control enzymatic
activities and cell fate [28]. Despite the view that cFLIP
requires procaspase-8 for binding to complex II, cFLIP
proteins may also act as control checkpoints of DED filament assembly [51]. Apart from TNF receptors, cFLIP
also protects by inhibiting the formation of the complexII-like cell death-inducing platform that other immune
receptors, including TLRs, can induce [52].
Heterodimerization with cFLIP is known to not only
prevent auto-processing of caspase-8 for apoptotic

function but also result in a limited and selective substrate specificity initiating diverse signaling outcomes[53–55]. Besides inhibiting caspase-8-mediated apoptosis, an essential role for cFLIP in regulating the life/
death decisions is the induction of NF-κB [56–58]. As a
caspase-8 substrate for pro-survival functions, procaspase-cleaved DED-containing fragments of cFLIP can
induce NF-κB activation via interacting with the IκBkinase (IκK) complex by binding to IκKγ [13, 59, 60].
Thus, besides regulating the functional divergence of caspase-8 in apoptotic and non-apoptotic pathways, cFLIP
controls NF-κB activation by directly interacting withmultiple proteins upstream of the NF-κB activation pathway. These support cFLIP functions beyond cell death
regulation.
The recent study exploring the molecular processes ofglaucoma linked cFLIP to cell type-dependent differencesin the biological activities of caspase-8, including apoptotic caspase-8 activity in RGCs and the non-apoptotic
function of caspase-8 in glial inflammatory responses[20]. As supported by previous studies [61, 62], the highexpression level of cFLIP in astroglia was considered a
critical factor for their different fate as opposed to RGCs
in glaucomatous samples [20]. While the RGCs in glaucomatous samples presented cleaved caspase-8, including
the p18 active enzyme, no apoptotic caspase-8 cleavage was detectable in the glaucomatous glia. Instead,Yang et al. Journal of Neuroinflammation(2024) 21:145

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Fig. 7 Western blot analysis of molecular responses. Western blots of astroglia proteins used phosphorylation site-specific antibodies to analyze phosphoRelA (p65) and phospho-RelB expression in transgenic and control mice with experimental glaucoma (G) compared to non-glaucomatous controls (C).
This analysis detected decreased phospho-RelA and increased RelB in ocular hypertensive GFAP/cFLIP compared to ocular hypertensive controls (cFLIPf/f).
Immunoblotting was repeated at least three times with samples collected from approximately 40 mice per group. Graphs present the fold change data(mean ± SD) from quantitative analysis of the β-actin-normalized band intensities in transgenic and control samples (*one-way ANOVA; p = 0.01)

astrocytes were detected to express the 55 kDa fulllength inactive proenzyme and the partially processed
p43 subunit that might result from heterodimerization ofprocaspase-8 with cFLIPL [50].
Cell death and inflammatory signals typically oppose
each other downstream of death receptor engagement, as
increasing apoptosis may be parallel to reduced inflammation, and caspase inhibition may result in enhanced
inflammatory cytokine production. This view is also consistent with the model for caspase-8 activity-dependent
cell fate decisions. While the high enzymatic activity of
caspase-8 promotes apoptosis in RGCs, in the absence offull proteolytic cleavage, the catalytic activity of caspase-8
signals cell survival and inflammation in astroglia [50].

In the present study, immunoblotting of astroglia proteins detected persisting p55 procaspase with increased
caspase-8 cleavage products, including p43 and p18, incFLIP-deleted samples. The detected cleavage products
might result from intra- and interdimeric self-cleavage of
caspase-8 in the lack of cFLIP [63]. Nevertheless, despite
increased self-processing of caspase-8, deletion of cFLIP
in astroglia could reduce proinflammatory responses inexperimental glaucoma while maintaining astroglia survival. Contrasting studies suggest cFLIP functioning as adual suppressor of apoptosis and inflammation; [64, 65]
however, differences in cell type, caspase activity, diseasemodel, and experimental paradigm make the comparisons difficult. As discussed below, additional molecularYang et al. Journal of Neuroinflammation(2024) 21:145

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Fig. 8 RGC counts. (A) Counting of the RBPMS-labeled RGCs (green) in retinal whole mounts detected a greater number of remaining RGCs in ocular
hypertensive GFAP/cFLIP and GFAP/cFLIPL compared to ocular hypertensive controls (cFLIPf/f and cFLIPLf/f), corresponding to approximately 36 and 39%
protection against RGC loss in experimental glaucoma. White boxed areas are shown in higher magnification (scale bar, 200 μm). (B) Graphs show thepercentage loss in RGC counts in different groups (*one-way ANOVA; p < 0.001). The data (mean ± SD) represent a minimum of 16 mice per groupalterations detected in the current study allow alternative
conclusions to be reached.
Alterations in inflammatory responses of astroglia with
cFLIP deletion

The molecular outcomes of this study indicated a
reduced inflammatory response to ocular hypertension with cFLIP-deletion in astroglia. Widespread glial
responses during glaucomatous neurodegeneration portray a continuum of multiple activation states exhibiting
tissue region- and disease-stage-dependent alterations.
A spatial and temporal heterogeneity in glial reactivity embraces a board of morphological and molecularresponses. Like previous studies of experimental glaucoma, the present study detected morphological characteristics of reactive astroglia, including hypertrophy of
their cellular processes with increased GFAP immunolabeling. However, the quantitative parameters, including
the intensity and percentage coverage of GFAP immunolabeling, exhibited only subtle changes between the ocular hypertensive retinas of mice with or without astroglial
cFLIP or cFLIPL deletion. Although no significant alteration was detectable in these morphological features,
inflammatory responses of the glaucomatous astroglia
presented various molecular changes in GFAP/cFLIP and
GFAP/cFLIPL mice.
Multiplex immunoassays detected decreased titers ofmultiple proinflammatory cytokines in the ocular hypertensive GFAP/cFLIP and GFAP/cFLIPL retina and optic
nerve compared to ocular hypertensive controls. While
proinflammatory cytokines, such as IL1, IL2, IFNγ, andTNFα, presented decreased titers, anti-inflammatory
cytokines, including IL4 and IL10, were significantly

increased ocular hypertensive tissues with GFAP/cFLIP
or GFAP/cFLIPL deletion in astroglia. Similar to the cytokine profiles displayed by immunoassays, the molecular changes detected by NanoString-based analysis ofastroglia samples found a significant increase in the antiinflammatory cytokine response, including Il10, and asignificant decrease in the proinflammatory cytokines,
such as Il15 and Il22, with astroglial cFLIP deletion.
As discussed in detail below, the alterations detected inproinflammatory and anti-inflammatory molecules withastroglial cFLIP deletion were also parallel to decreased
RelA but increased RelB expression and activity. Thesemolecular outcomes may point to a regulatory mechanism to limit proinflammatory outcomes and prevent cell
death.
Additional molecules presenting altered expression
in GFAP/cFLIP samples included some gene targets of
NF-κB. For example, TNFα-induced protein-3 (Tnfaip3,
also called A20) was among the downregulated molecules in cFLIP-deleted astroglia. A20 is known to regulate
TNFα-mediated inflammation and apoptosis [66–68].
The decreased expression of Tnfaip3, which was previously found to be an epigenetic target in glaucomatoushuman eyes [4], might be secondary to inhibited TNFR
signaling and inflammatory outcomes.
In addition, cFLIP-deleted astroglia upregulated several complement components. Previous studies havedemonstrated the complement system as a potent modulator and effector of diverse glial functions in glaucomatous neurodegeneration [27, 69]. Astrocytes normally
maintaining the homeostasis of the central nervous
system are well-equipped to receive and send complement-related signals. Many of the molecules involved inYang et al. Journal of Neuroinflammation(2024) 21:145

the complement activation pathway, including various
receptors and regulators, are expressed by astrocytes.
They play critical roles in their crosstalk with microglia,
besides tissue clearance, protection, or damage whendysregulated. Evidently, the dynamic shift between different activation states and functions of astrocytes in theearly or late stages of glaucoma includes complement
components [70]. For example, transgenic targeting ofcomplement C3 delayed the progression of neurodegeneration in mice with hereditary glaucoma; [71] however,C3 induced neuroprotective functions of astrocytes at
early disease stages [72].
Alterations in NF-κB expression of astroglia with cFLIP
deletion

A remarkable observation from the NanoString-based
molecular analysis was decreased RelA but increased
RelB expression in cFLIP-deleted astroglia. This observation was also matched with decreased phospho-RelA
and increased phospho-RelB expression in cFLIP-deleted
astroglia. The five structurally related monomers of the
NF-κB signaling system, including RelA (p65), RelB,
NF-κB1 (p105/p50), NF-κB2 (p100/p52), and c-Rel,mediates dimerization, interaction with IκBs, and nuclear
translocation for transcription of several inflammatory,
developmental, and survival genes. Various hetero- or
homodimers mediate transcription of target genes bybinding to a specific DNA element, κB enhancer. Different NF-κB dimeric complexes are expressed in a cell
type- and stimulus-specific manner. NF-κB family members present sequence homology in their DNA binding
and dimerization domains and participate in transcriptional activation of hundreds of genes; however, despite
their close relation in sequence and function, RelA and
RelB also display distinct effects on gene expression [73,74]. Multiple studies have demonstrated that they modulate each other’s function and repress NF-κB-dependent
gene expression [75, 76]. Target genes for the transcription mediated through RelA: p50 and cRel: p50 heterodimers in the canonical pathway are mainly involved ininnate immunity. Although inflammatory responses typically occur through activation of the canonical pathway,
RelB, with its DNA-binding partners p50 and p52, plays
its specific roles along the non-canonical NF-κB pathway. The new synthesis of RelB allows for the generationof RelB: p52 in this alternative pathway, which is insensitive to IκB control. The non-canonical NF-κB pathway
appears to be a supplementary signaling axis that cooperates with the canonical pathway and governs the gene
regulation predominantly involved in lymphoid organ
development and adaptive immunity [77–80]. Previous
studies have shown cross-regulation between RelA and
RelB on TNFα-induced NF-κB target gene expression.
RelA regulates the transcription of RelB [81], promotes

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the generation of RelB/p52 dimers [82], and plays a regulatory role to diminish RelB activity for selective target
gene expression in response to TNFα [76]. A more recent
study supporting a cross-talk between the canonical andnon-canonical NF-κB pathways showed that reductionin cellular RelB levels led to dysregulation of the NF-κB
pathway, promoting a shift towards proinflammation due
to the removal of the RelB-mediated inhibition of RelAinduced proinflammatory transcriptional activity [83].
Unlike RelA, RelB remained understudied in glaucoma
research. This is partly because TNFα signaling, a prominent component of the astroglia-driven neuroinflammation in the glaucomatous retina, appears to mainly induce
RelA: p50 dimers [84]. Interestingly, molecular analysis
detected decreased RelA expression and phosphorylated
RelA with cFLIP-deletion in ocular hypertensive astroglia. On the contrary, RelB expression and phospho-RelB
were increased, supporting an upregulated transcriptional function of RelB [85, 86] in ocular hypertensive
GFAP/cFLIP compared to ocular hypertensive controls.
These findings indicate the non-canonical pathway activated in ocular hypertensive samples of cFLIP-deleted
astroglia. Since the canonical and non-canonical NF-κB
pathways may impact each other, we wonder whether
the increased RelB expression we detected could be compensatory to decreased RelA or an independent response
for further repressing the proinflammatory activation. In
support of this view, RelB knock-out mice were reported
to suffer multi-organ inflammation [87], and overexpression of RelB in LPS-stimulated macrophages suppressed proinflammatory TNFα production [88]. In more
recent studies, increased expression of RelB in astrocytes
induced immune tolerance in experimental neuroinflammation due to silencing the proinflammatory cytokine
genes after RelB: p50 binding to DNA [89, 90].
Thus, the molecular data pointed to a regulatory mechanism to diminish RelA and promote RelB for selective
expression of NF-κB target genes in cFLIP-deleted astroglia. This molecular regulation seems to limit the expression of a subset of RelA-responsive genes, including
proinflammatory cytokines. Respecting the cell type-specific differences in NF-κB activation and function [91], abetter understanding of the cell type-specificity of theseobservations awaits further investigation. As discussed
below, the changed expression pattern of RelA and RelB
may also have implications for astroglia survival of thecFLIP deletion.
Transgenic effects on survival of astroglia with cFLIP
deletion

Concerning the critical role of caspase-8/cFLIP interactions in cell fate, one possibility after cFLIP deletion could
be the signal shift toward caspase-8-initiated apoptosis.
Although caspase-8:cFLIPL heterodimers may possess

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Fig. 9 Modeling of the experimental outcomes. While the longer isoform of cFLIP (cFLIPL) is composed of tandem death effector domains (DEDs) and acatalytically inactive caspase-like domain, the short isoform (c-FLIPR) is a truncated version. As a protease-deficient caspase-8 homolog, the heterodimerof cFLIP with caspase-8 prevents autocleavage-mediated apoptotic activation requiring homodimerization (as detected in glaucomatous RGCs). Besides
inhibiting caspase-8-mediated apoptosis, high cFLIP expression also plays a critical role in regulating NF-κB-mediated inflammatory outcomes (as detected in glaucomatous astroglia). The findings of this study support that deletion of cFLIP, or cFLIPL, in astroglia can reduce proinflammatory responses andinflammatory injury to RGCs in experimental glaucoma. Accompanying molecular alterations also imply that a transcriptional autoregulatory response,
including induction of RelB, reinforces pro-survival, despite increased caspase-8 cleavage in cFLIP-deleted astroglia

restricted protease activity, and the resultant cleavage
product, p43, can cleave some downstream effectors of
apoptosis, short isoform can terminate the formation oftandem homo-oligomers propagating cleavage-mediatedcaspase-8 activation [28]. As modeled in Fig. 9, in themice with astroglial cFLIPL deletion, intact cFLIPR (thatdoes not have a caspase-like domain, but DED, common for both isoforms) can prevent procaspase-8 homooligomer assembly needed for the full cleavage. This

study detected increased caspase-8 cleavage; however,no change was detectable when apoptotic cell death wasanalyzed in astroglia. Not only GFAP/cFLIPL but also
GFAP/cFLIP, lacking both isoforms, presented no induction of astroglial apoptosis at least at 12 weeks of ocular
hypertension.
There may be different explanations for protection
against apoptosis, which warrants additional studies
to elucidate further. Most importantly, the molecularYang et al. Journal of Neuroinflammation(2024) 21:145

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alterations detected seemed decisive for cell survival.
While decreased RelA might be associated with reduced
inflammatory response, increased RelB might be sufficient to prevent apoptosis since it is known to promoteseveral anti-apoptotic gene targets [91, 92]. Previous RelB
mutant studies demonstrated the contribution of RelB
to anti-apoptotic signaling and cell survival during thymocyte maturation [93]. The anti-apoptotic gene expression might persist despite RelB-mediated silencing of thecytokine response [76, 94]. Studies using RelB-deficient
cells showed that the selective target gene expression by
RelB includes an abundance of anti-apoptotic molecules,
including Bcl-2 and Bcl-xL, and inhibitor of apoptosis
proteins (IAP), in addition to the regulators of cell cycletransition and proliferation, such as c-Myc and cyclins[95, 96]. Besides inhibiting cell death, the IAP controls
ubiquitin-dependent pathways that regulate RIPK1/
NF-κB activation, thereby modulating inflammation [97].
Moreover, RelB might contribute to cellular protection by
inducing an antioxidant response [98, 99]. The decreased
nos2 detected in cFLIP-deleted astroglia seems consistent
with such an antioxidant response.
Additionally, caspase-8 auto-processing through a
combined series of intra- and interdimeric cleavages is
accelerated with increasing ligand binding for receptor
signaling [97]. Therefore, decreased production of specific proapoptotic ligands, such as proinflammatory cytokine TNFα, and decreased stimulus strength of the death
receptor might cause weakness in receptor signaling,
contributing to the control of apoptosis. In similar interest to RelB, A20, also decreased in expression in cFLIPdeleted astroglia, is known to suppress both classical andalternative NF-κB and block the expression of specificanti-apoptotic genes [98].
Thus, the immunomodulatory transgenic effect withastroglial cFLIP deletion did not shift the experimental
paradigm to caspase-8-mediated cell death; however, atranscriptional autoregulatory response, including induction of RelB, was considered to reinforce pro-survival.
Ongoing studies through a more chronic period of ocular
hypertension are expected to provide further information to determine whether cell survival in cFLIP-deleted
astroglia endures long-term. It should also be clarified
whether apoptosis, if induced long-term, would specifically affect astroglia’s proinflammatory/neurotoxic
states because these cells would be primed for caspase-8
expression and activities through TNFR, TLR signaling,or the inflammasome activation in glaucoma.

modulation of cFLIP expression in astroglia activateda transcriptional autoregulatory response, dampening
RelA but boosting RelB for selective expression of NF-κB
target genes, reinforcing cell survival in cFLIP-deleted
astroglia while limiting proinflammatory responses. In
light of the presented findings, targeting the caspase-8/
cFLIP interaction in astroglia may offer a treatment strategy against NF-κB-regulated neuroinflammation andinflammatory injury to neurons. Concerning the direct
cytotoxicity of astroglia-produced proinflammatory cytokines, including TNFα [8–10], to RGCs in glaucoma anda prolonged amplification cycle of the glia-driven neurodegenerative inflammation stimulated by downstream
NF-κB activation [6], such an astroglia-targeting immunomodulatory approach can provide long-term protection to these precious neurons. Continuing studies for
improved molecular understanding of neuroinflammation will be the key to opening the way to a therapeutic
approach by immunomodulation in glaucoma and otherneuroinflammatory neurodegenerative diseases.

Conclusions
In summary, the findings of this study accumulated
supportive data for the astroglial cFLIP deletion to prevent/reverse the neurodegenerative proinflammatory
outcomes of experimental glaucoma. The transgenic

Funding
This study was supported by a research grant from the National Eye Institute,
Bethesda, MD (R01 EY033359, GT) and departmental grants from the National
Eye Institute (P30 EY019007) and Research to Prevent Blindness Inc. (NewYork, NY). Images were collected in the Confocal and Specialized Microscopy
Shared Resource of the Herbert Irving Comprehensive Cancer Center atAbbreviations
Cflar	Caspase-8 FADD-like apoptosis regulator
cFLIP	FLICE-like inhibitory protein
DED	Death effector domain
GFAP	Glial fibrillary acidic protein
IκKβ	Inhibitor of kappaB kinase-subunit beta
IFN-γ	Interferon-gammaIL	Interleukin, NF-κB, NIK, NF-κB-inducing kinase, nuclear
factor-kappaB
RELA/B	v-Rel avian reticuloendotheliosis viral oncogene homolog A/B
RIPK	Receptor-interacting serine/threonine-protein kinase 1,RGC, retinal
ganglion cell
RPBMS	RNA-binding protein with multiple splicing
TLR	toll-like receptor
TNF-α	Tumor necrosis factor-alpha
TNFR	Tumor necrosis factor receptor
TUNEL	Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End
Labeling

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03141-4.
Supplementary Material 1
Acknowledgements
Not applicable.
Author contributions
X.Y. and Q.Z. were involved in the generation and genotyping of mouse lines,
in vivo experimentation, and data curation; G.İ. and O.İ. were involved in image
and data analysis; L.C. was engaged in the generation of the cFLIPLf/f mouseline; G.T. was involved in conception and supervision of the study and writingof the manuscript.

Yang et al. Journal of Neuroinflammation(2024) 21:145

Columbia University, supported by a grant from the National Institute of
Health (P30 CA013696). The presented work also utilized the resources ofColumbia University’s Human Immune Monitoring Core.
Data availability
The generated and analyzed data during this study are included in this article,
and methodological details and materials will be available to interested
parties upon request.

Declarations
Ethics approval and consent to participate
All experiments with mice were approved by the Institutional Animal Careand Use Committee of Columbia University. The procedures complied with
the American Veterinary Medical Association Guidelines and the tenets of the
ARVO statement for using animals in ophthalmic and vision research.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 5 April 2024 / Accepted: 24 May 2024

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