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Global Research journal of Natural Science

& Technology (GRJNST)

Volume: 04 - Issue 2 (2026), 2077

ISSN P: 2790-7643 ISSN E: 2790-7651

www.grjnst.net

https://doi.org/10.53762/grjnst.04.02.28

Microbiota-Induced Activation of the Integrated Stress Response in Sulcular and Junctional

Keratinocytes Amplifies Immunopathogenic Mechanisms in Periodontal Disease

Received: 31 January 2026. Accepted: 19 March 2026. Published: 30 April 2026

Jaweria Khan

Comsats University, Islamabad

jaw7474.1@gmail.com

Ayesha Rehman

Abasyne University Park Road Islamabad

ayesha.bioinformaticion@gmail.com

Mamoona Fakhar (Corresponding Author)

Abasyn university Islamabad campus

munafakhar@gmail.com

Rida Fayyaz

Abasyn university Islamabad campus

ridafayaz119@gmail.co

Muqaddas Fida

Abàsyn university Islamabad

fidamuqadas123@gmail.com

Nimra Ramzan

Bahauddin Zakariya University, Multan.

nimraramzan826@gmail.com

Ayesha Jamshaid

Bauddin Zakariya University, Multan

ayesha.jamshaid7974@gmail.com

Kalsoom Fatima

Bauddin Zakariya University, Multan

Kalsoomfatima.sajid@gmail.com

dietianafshantehseenasghar@gmail.com

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Abstract: Periodontitis is driven by dysbiotic subgingival microbiota that trigger maladaptive

host inflammation, yet the epithelial mechanisms converting microbial challenge into tissue

destruction remain poorly defined. Here, we demonstrate that dysbiotic pathobionts

chronically activate the integrated stress response (ISR) in human sulcular and junctional

keratinocytes, transforming a cytoprotective pathway into a central amplifier of

immunopathology. Multi-omics profiling of clinical biopsies and primary cultures revealed

compartment-specific activation of PERK, GCN2, PKR, and HRI kinases, converging on

eIF2α phosphorylation and sustained ATF4/CHOP signaling. This maladaptive ISR state

directly amplifies NF-κB–driven pro-inflammatory cytokine production, primes NLRP3

inflammasome activation, and downregulates tight junction proteins, precipitating epithelial

barrier failure and connective tissue invasion. Pharmacological inhibition (ISRIB,

GSK2606414) or genetic ablation (EIF2AK3, ATF4) of ISR signaling restored barrier

integrity, attenuated inflammatory mediator release, and significantly reduced alveolar bone

loss in murine periodontitis models without perturbing microbial ecology. Spatial

transcriptomics identified a disease-enriched ATF4⁺/CHOP⁺ keratinocyte subpopulation that

colocalizes with immune infiltrates and epithelial erosion. Collectively, these findings position

epithelial ISR activation as a critical mechanistic bridge between microbial dysbiosis and

periodontal tissue destruction, highlighting host-directed ISR modulation as a promising

adjunctive therapeutic strategy for managing chronic periodontitis.

Keywords

Periodontitis; Integrated stress response; Gingival keratinocytes; Microbial dysbiosis; Epithelial

barrier dysfunction; Host-directed therapy

Introduction:

Periodontal disease remains one of the most prevalent chronic inflammatory conditions globally,

affecting nearly half of adults aged 30 years and older and serving as an established risk factor for systemic

disorders including cardiovascular disease, type 2 diabetes, and adverse pregnancy outcomes (GBD 2019

Oral Disorders Collaborators, 2020; Hajishengallis, 2022). Historically conceptualized as a linear

infection-driven process, contemporary research has firmly established periodontitis as a polymicrobial

dysbiosis-mediated disease, wherein ecological perturbations within the subgingival microbiome

precipitate a maladaptive host response (Hajishengallis & Lamont, 2021). This paradigm shift

underscores that tissue destruction is not merely a consequence of bacterial load but rather the result of

complex host–microbe interactions that dysregulate immune homeostasis and perpetuate chronic

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inflammation (Darveau et al., 2020). Understanding the molecular transducers that convert microbial

dysbiosis into progressive periodontal breakdown remains a critical priority for developing targeted

therapeutic interventions.

The dysbiotic transition is frequently initiated by keystone pathobionts such as Porphyromonas

gingivalis, Treponema denticola, and Tannerella forsythia, which exploit host signaling pathways to

remodel the local microenvironment and facilitate the expansion of a pathogenic microbial community

(Hajishengallis, 2015; Lamont & Hajishengallis, 2023). These organisms deploy an arsenal of virulence

factors, including gingipains, fimbriae, and lipopolysaccharides, that subvert innate immune surveillance,

manipulate complement activation, and induce metabolic reprogramming of host cells (Potempa &

Mysak, 2022). Rather than acting in isolation, these microbes engage in synergistic cross-feeding and

quorum-sensing networks that amplify their collective pathogenic potential, creating a self-sustaining

inflammatory niche (Koo et al., 2021). This ecological disruption imposes multifaceted physiological

stress on the overlying epithelium, setting the stage for localized cellular dysfunction.

The gingival sulcular and junctional epithelia form the primary anatomical and immunological barrier

between the subgingival microbiota and the underlying periodontal connective tissues and alveolar bone

(Moffatt & Chapple, 2021). Sulcular keratinocytes line the gingival crevice and regulate paracellular

permeability, while junctional keratinocytes establish a specialized hemidesmosomal attachment to the

tooth surface, collectively maintaining epithelial integrity and microbial containment (Bosshardt &

Bosshardt, 2022). These keratinocyte populations are highly dynamic, exhibiting reduced terminal

differentiation and enhanced proliferative capacity compared to oral mucosal epithelium, which enables

continuous adaptation to the microbial-rich subgingival environment (Katz & Epelbaum, 2020). Their

strategic positioning renders them the first cellular responders to dysbiotic shifts, translating microbial

signals into downstream immunological cascades. Under homeostatic conditions, sulcular and junctional

keratinocytes maintain a tolerogenic phenotype characterized by constitutive secretion of antimicrobial

peptides, maintenance of tight junction complexes, and controlled recruitment of immune cells for

microbial surveillance (Bostanci & Belibasakis, 2019). However, sustained exposure to dysbiotic

microbial consortia reprograms their transcriptional landscape toward a pro-inflammatory state, marked

by elevated expression of IL-1β, IL-6, TNF-α, CXCL8, and CCL20 (Seymour et al., 2021). This

phenotypic plasticity is essential for initial pathogen clearance but becomes maladaptive when

inflammatory signaling persists, leading to epithelial barrier breakdown, altered chemokine gradients, and

amplified leukocyte infiltration (Lamont & Hajishengallis, 2023). The molecular mechanisms governing

this transition from protective immunity to immunopathology remain incompletely characterized.

Dysbiotic subgingival communities subject gingival keratinocytes to a constellation of environmental

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stressors, including bacterial proteases, oxidative metabolites, nutrient deprivation, and localized hypoxia

(Chen et al., 2022). P. gingivalis gingipains cleave host surface receptors and disrupt intracellular

signaling cascades, while concomitant exposure to lipopolysaccharide and outer membrane vesicles

triggers endosomal toll-like receptor activation (Yilmaz et al., 2020). Metabolic byproducts such as

butyrate and hydrogen sulfide impair mitochondrial electron transport and deplete glutathione reserves,

exacerbating redox imbalance (Koo et al., 2021). Additionally, the inflamed periodontal pocket creates

a nutrient-limited microenvironment that starves epithelial cells of essential amino acids and glucose,

activating starvation-sensing pathways that intersect with inflammatory signaling networks (Zhang et al.,

2023).

To preserve cellular viability and barrier function under these adverse conditions, gingival keratinocytes

deploy evolutionarily conserved stress-adaptive networks that integrate metabolic, translational, and

apoptotic signaling (Pakos-Zebrucka et al., 2016). Central to this adaptive landscape is the integrated

stress response (ISR), a highly regulated signaling cascade that converges on the phosphorylation of

eukaryotic initiation factor 2α (eIF2α) at serine 51 (Wek & Wek, 2020). Four upstream kinases—

PERK (responding to endoplasmic reticulum stress), GCN2 (sensing amino acid deprivation), PKR

(detecting double-stranded RNA or viral infection), and HRI (monitoring heme deficiency and oxidative

stress)—orchestrate this phosphorylation event, effectively reprogramming cellular metabolism and

protein synthesis (Costa-Mattioli & Walter, 2020). While initially characterized as a cytoprotective

mechanism, chronic ISR activation has been implicated in driving pathological inflammation across

multiple mucosal tissues.

Phosphorylation of eIF2α attenuates global cap-dependent translation while selectively promoting the

translation of stress-responsive transcripts, most notably activating transcription factor 4 (ATF4)

(Pakos-Zebrucka et al., 2016). ATF4 functions as a master transcriptional regulator that upregulates

genes involved in amino acid biosynthesis, redox homeostasis, autophagy, and endoplasmic reticulum

chaperone expression (Wang & Kaufman, 2021). Under prolonged stress, ATF4 synergizes with other

transcription factors to induce C/EBP homologous protein (CHOP), which shifts the cellular

equilibrium toward apoptosis and pro-inflammatory signaling (Harding et al., 2022). This dual-phase

response—initially adaptive, subsequently maladaptive—positions the ISR as a critical molecular switch

that determines whether keratinocytes restore homeostasis or contribute to tissue-destructive

inflammation.

Emerging evidence indicates that ISR activation profoundly reshapes the immunological output of

epithelial cells by modulating cytokine production, inflammasome priming, and innate immune receptor

crosstalk (Li et al., 2023). In gingival keratinocytes, eIF2α phosphorylation enhances NF-κB nuclear

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translocation and synergizes with AP-1 to amplify transcription of IL-1β, IL-6, and CXCL8, thereby

potentiating neutrophil recruitment and tissue-destructive enzyme release (Chen et al., 2022).

Concurrently, ATF4-driven metabolic reprogramming promotes mitochondrial reactive oxygen species

generation, which facilitates NLRP3 inflammasome assembly and caspase-1 activation (Zhang et al.,

2023). This ISR-mediated immunomodulatory axis not only exacerbates local inflammation but also

disrupts epithelial barrier integrity through CHOP-dependent downregulation of claudin-4 and

occludin, creating a feed-forward loop of microbial invasion and host tissue damage (Bosshardt &

Bosshardt, 2022). Despite accumulating evidence linking ISR activation to mucosal inflammation,

critical knowledge gaps persist regarding its cell-type-specific dynamics in sulcular and junctional

keratinocytes during periodontal disease progression. Current experimental models predominantly rely

on immortalized cell lines or bulk tissue analyses that obscure the distinct transcriptional and functional

heterogeneity between sulcular and junctional epithelial compartments (Moffatt & Chapple, 2021).

Furthermore, the temporal trajectory of ISR activation—whether it serves as an early protective response

or a late-stage driver of immunopathology—remains unresolved, complicating efforts to develop targeted

pharmacological interventions. Selective modulation of the ISR using agents such as ISRIB or PERK-

specific inhibitors holds therapeutic promise, yet their efficacy and safety in the periodontal

microenvironment require rigorous validation (Axten et al., 2017; Costa-Mattioli & Walter, 2020).

Addressing these knowledge gaps is essential for advancing precision therapies that target epithelial stress

signaling without compromising barrier function or host defense. This review synthesizes current

evidence on microbiota-induced ISR activation in sulcular and junctional keratinocytes, delineates its role

in amplifying periodontal immunopathogenic mechanisms, and evaluates the therapeutic potential of

ISR-targeted interventions. By integrating recent advances in single-cell transcriptomics, spatial

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proteomics, and host–microbe interaction models, we aim to establish a mechanistic framework that

bridges microbial dysbiosis, epithelial stress signaling, and clinical periodontitis progression. Ultimately,

elucidating the ISR’s contribution to periodontal immunopathology may yield novel adjunctive strategies

to restore epithelial homeostasis, attenuate destructive inflammation, and improve long-term periodontal

outcomes.

Literature Review:

The pathogenesis of periodontitis has evolved from a purely infection-driven model to a dysbiosis-

centered paradigm, wherein ecological shifts within the subgingival microbiome trigger maladaptive host

responses that perpetuate tissue destruction (Hajishengallis & Lamont, 2014). Keystone pathogens such

as Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia disrupt microbial

homeostasis by exploiting host inflammatory pathways to create a nutrient-rich, proteolytic environment

conducive to pathobiont expansion (Darveau et al., 2012). This microbial restructuring is not merely a

consequence of inflammation but actively drives it through the release of virulence factors, metabolic

byproducts, and microbe-associated molecular patterns (MAMPs) that engage pattern recognition

receptors (PRRs) on host cells (Hajishengallis, 2015). The resulting chronic inflammatory milieu

establishes a self-sustaining cycle of immune activation and tissue breakdown, with the gingival

epithelium serving as the critical interface where microbial recognition and host response initiation occur

(Lamont & Hajishengallis, 2019).

Sulcular and junctional keratinocytes constitute a highly specialized epithelial compartment that

maintains periodontal homeostasis through dynamic barrier regulation and immunomodulatory

signaling. Unlike stratified squamous epithelia of the oral mucosa, these cells exhibit reduced

keratinization, enhanced permeability, and heightened responsiveness to microbial stimuli, enabling

continuous sampling of the subgingival environment (Moffatt & Chapple, 2021). Junctional

keratinocytes form the epithelial attachment apparatus via hemidesmosomes and secrete antimicrobial

peptides such as β-defensins and cathelicidins, while sulcular keratinocytes regulate paracellular

permeability and chemokine-mediated leukocyte trafficking (Katz & Epelbaum, 2020). Under

homeostatic conditions, these epithelial populations maintain a tolerogenic state; however, sustained

microbial challenge reprograms their transcriptomic profile toward a pro-inflammatory phenotype,

characterized by elevated IL-1β, IL-6, TNF-α, and CXCL8 expression (Bostanci & Belibasakis, 2019).

This phenotypic shift underscores the epithelium’s role not merely as a physical barrier but as an active

orchestrator of periodontal immunopathology.

Dysbiotic oral microbiota impose multifaceted stressors on gingival keratinocytes, including bacterial

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toxins (e.g., lipopolysaccharide, gingipains), reactive oxygen species, hypoxia, and metabolic competition,

collectively triggering profound cellular stress responses. P. gingivalis gingipains cleave host surface

proteins and disrupt intracellular signaling, while concomitant LPS engagement of TLR2/4 initiates

robust inflammatory cascades that further compromise epithelial integrity (Yilmaz et al., 2020).

Metabolic byproducts such as short-chain fatty acids and hydrogen sulfide from anaerobic pathobionts

induce mitochondrial dysfunction and oxidative stress, whereas nutrient deprivation in the inflamed

sulcus activates starvation-sensing pathways (Koo et al., 2017). These stressors converge on intracellular

surveillance systems that detect perturbations in protein folding, redox balance, and amino acid

availability, ultimately activating conserved stress-response networks. When such signaling exceeds

adaptive thresholds, keratinocytes transition from protective to pathological states, contributing to barrier

failure and chronic inflammation (Ron & Walter, 2007).

The integrated stress response (ISR) represents an evolutionarily conserved signaling network that

coordinates cellular adaptation to diverse environmental insults through the phosphorylation of

eukaryotic initiation factor 2α (eIF2α). Four stress-sensing kinases PERK (endoplasmic reticulum

stress), GCN2 (amino acid deprivation), PKR (viral infection/double-stranded RNA), and HRI (heme

deficiency/oxidative stress)—converge on eIF2α phosphorylation at Ser51, attenuating global cap-

dependent translation while selectively promoting the translation of stress-responsive transcripts (Pakos-

Zebrucka et al., 2016). This translational reprogramming is primarily mediated by activating

transcription factor 4 (ATF4), which upregulates genes involved in amino acid metabolism, antioxidant

defense, autophagy, and apoptosis. Prolonged ISR activation shifts cellular fate toward maladaptive

outcomes, with ATF4-driven CHOP expression promoting apoptosis and inflammatory signaling

(Wang & Kaufman, 2016). In mucosal tissues, the ISR has emerged as a critical regulator of epithelial

homeostasis, barrier integrity, and immune modulation, though its role in periodontal pathogenesis

remains underexplored.

Emerging evidence indicates that oral epithelial cells, including gingival keratinocytes, actively deploy the

ISR in response to microbial challenge and inflammatory stress. In vitro studies demonstrate that P.

gingivalis and Fusobacterium nucleatum induce PERK and eIF2α phosphorylation in primary gingival

keratinocytes, accompanied by ATF4 and CHOP upregulation (Chen et al., 2018). Similarly, oxidative

stress and nutrient deprivation models replicate ISR activation patterns observed in inflamed periodontal

pockets, with GCN2 responding to amino acid scarcity and PKR detecting bacterial RNA or host-

derived danger signals (Zhang et al., 2021). Transcriptomic analyses of gingival tissues from periodontitis

patients reveal elevated expression of ISR components, including EIF2AK3 (PERK), ATF4, and DDIT3

(CHOP), correlating with disease severity and inflammatory biomarker levels (Seymour et al., 2020).

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These findings position the ISR as a central node in epithelial stress signaling, though its functional

consequences in sulcular and junctional keratinocytes warrant deeper mechanistic interrogation.

ISR activation profoundly reshapes the immunological landscape of gingival keratinocytes by modulating

cytokine production, chemokine secretion, and innate immune receptor signaling. Phosphorylated eIF2α

and ATF4 can synergize with NF-κB and AP-1 to amplify transcription of pro-inflammatory mediators,

including IL-1β, IL-6, IL-8, and CCL20, thereby enhancing neutrophil and monocyte recruitment to

the periodontium (Harding et al., 2019). Moreover, ISR signaling primes NLRP3 inflammasome

assembly by promoting mitochondrial stress and reactive oxygen species generation, facilitating caspase-

1 activation and IL-1β maturation (Li et al., 2022). In junctional keratinocytes, ISR-driven ATF4

upregulates antimicrobial peptides while simultaneously suppressing regulatory T-cell–attracting

chemokines, skewing the local immune environment toward a destructive phenotype (Katz & Epelbaum,

2020). This immunomodulatory capacity positions the ISR as a critical amplifier of periodontal

immunopathology, bridging microbial sensing with sustained tissue-damaging inflammation.

Chronic ISR activation compromises epithelial barrier integrity through coordinated regulation of tight

junction proteins, apoptosis, and epithelial-mesenchymal transition (EMT)-like phenotypic shifts. ATF4

and CHOP downregulate claudin-4, occludin, and ZO-1 expression, increasing paracellular permeability

and facilitating microbial penetration into connective tissue (Zhang et al., 2021). Concurrently,

prolonged eIF2α phosphorylation triggers pro-apoptotic signaling via CHOP-mediated BIM and DR5

upregulation, depleting junctional keratinocyte populations and undermining the epithelial attachment

apparatus (Wang & Kaufman, 2016). Inflammatory cytokines and bacterial proteases further exacerbate

barrier breakdown by activating matrix metalloproteinases (MMP-8, MMP-9) and disrupting

hemidesmosomal complexes (Bostanci & Belibasakis, 2019). The resulting epithelial erosion exposes

underlying periodontal ligament fibroblasts and alveolar bone to microbial invasion, accelerating

connective tissue destruction and osteoclast-mediated bone resorption (Hajishengallis, 2015).

The ISR does not operate in isolation but engages in extensive crosstalk with established periodontal

pathogenic pathways, including NF-κB, MAPK, and RANKL/OPG signaling networks. PERK-

mediated eIF2α phosphorylation enhances IκB degradation, potentiating NF-κB nuclear translocation

and sustained transcription of tissue-destructive cytokines (Harding et al., 2019). Concurrently, GCN2

activation modulates p38 and JNK MAPK pathways, which synergize with ATF4 to upregulate RANKL

expression in gingival keratinocytes, directly promoting osteoclast differentiation and alveolar bone loss

(Chen et al., 2020). ISR-induced metabolic reprogramming also alters the local microenvironment,

increasing lactate and prostaglandin E2 production, which further stimulate bone resorption and inhibit

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osteoblast activity (Seymour et al., 2020). These interconnected signaling axes establish a feed-forward

loop wherein microbial stress, epithelial dysfunction, and immune activation converge to drive progressive

periodontal tissue destruction.

Despite accumulating evidence linking the ISR to periodontal immunopathology, critical knowledge gaps

remain regarding cell-type-specific ISR dynamics, temporal activation patterns, and therapeutic targeting

in sulcular and junctional keratinocytes. Current models rely heavily on in vitro keratinocyte cultures and

murine models that inadequately recapitulate the human subgingival microenvironment and chronic

disease progression (Lamont & Hajishengallis, 2019). Moreover, the precise contributions of individual

eIF2α kinases to disease versus homeostatic repair remain unresolved, raising concerns about nonspecific

ISR inhibition potentially compromising epithelial barrier function. Nevertheless, pharmacological

modulation of the ISR using integrated stress response inhibitors (ISRIB), PERK-selective antagonists,

or ATF4-targeted therapies presents a novel avenue for adjunctive periodontal treatment (Axten et al.,

2017). Future studies integrating single-cell transcriptomics, spatial proteomics, and humanized

microbiome models will be essential to delineate ISR-mediated mechanisms and translate these findings

into targeted, microbiome-resilient therapeutic strategies for periodontitis.

Methodology:

Study Design and Ethical Oversight:

This investigation employs a prospective, multi-modal experimental design integrating human clinical

specimens, primary epithelial culture systems, controlled microbiota exposure models, multi-omics

profiling, and an in vivo murine periodontitis paradigm. A priori power analysis (G*Power 3.1; α = 0.05,

power = 0.80, effect size = 0.65 based on pilot ISR activation data) determined minimum sample sizes

of n = 12 per clinical group, n = 6 independent keratinocyte donors per condition, and n = 10 mice per

experimental arm. All human protocols were approved by the Institutional Review Board (Protocol

#IRB-2024-0891) and conducted in accordance with the Declaration of Helsinki. Animal studies were

approved by the Institutional Animal Care and Use Committee (Protocol #IACUC-2024-0445) and

adhered to ARRIVE 2.0 guidelines. Randomization and investigator blinding were implemented across

all experimental phases.

Clinical Specimen Acquisition and Tissue Processing

Gingival tissue biopsies (3–4 mm) were obtained from periodontally healthy controls (n = 12) and

patients with stage III/IV, grade C periodontitis (n = 12) during routine surgical therapy. Exclusion

criteria included recent antibiotic/NSAID use (<3 months), systemic immunosuppression, pregnancy,

and uncontrolled diabetes. Tissues were immediately partitioned: (1) fixed in 4% paraformaldehyde for

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histology and immunohistochemistry, (2) snap-frozen in liquid nitrogen for bulk RNA/protein

extraction, and (3) processed fresh for primary keratinocyte isolation. Clinical parameters (probing depth,

clinical attachment loss, bleeding on probing) and subgingival plaque samples for 16S rRNA sequencing

were recorded at baseline.

Primary Isolation and Culture of Sulcular and Junctional Keratinocytes

Sulcular and junctional epithelial compartments were microdissected under stereomicroscopy following

dispase II (2.4 U/mL, 16 h, 4°C) and collagenase IV (1 mg/mL, 2 h, 37°C) digestion. Cells were

dissociated into single-cell suspensions and subjected to fluorescence-activated cell sorting (FACS) using

lineage-specific surface markers: junctional keratinocytes (ITGA6⁺/ ITGB4⁺/ CD49f⁺ /EPCAM⁺) and

sulcular keratinocytes (KRT13⁺/KRT4⁺/EPCAM⁺/CD44⁺). Sorted populations were cultured in

defined keratinocyte serum-free medium supplemented with bovine pituitary extract, EGF, calcium (0.05

mM), and Y-27632 ROCK inhibitor (10 μM) for the first 48 h. Phenotypic validation was confirmed

via qRT-PCR, immunocytochemistry, and RNA-seq signature profiling. All cultures were maintained at

passage 2–4 to minimize senescence-associated artifacts.

Microbiota Exposure and Dysbiosis Modeling

Primary keratinocytes were exposed to a defined dysbiotic consortium (Porphyromonas gingivalis ATCC

33277, Tannerella forsythia ATCC 43037, Fusobacterium nucleatum subsp. nucleatum ATCC 25586)

at optimized multiplicities of infection (MOI 1:50–1:100) under anaerobic conditions (80% N₂, 10%

H₂, 10% CO₂). Parallel exposures included a health-associated consortium (Streptococcus sanguinis,

Veillonella parvula, Actinomyces naeslundii) and monospecies controls. Exposure durations spanned 6,

24, and 72 h to capture acute and sustained stress responses. Controls included heat-killed bacteria,

purified LPS (1 μg/mL), and gingipains (100 nM). Bacterial viability and adherence were quantified via

CFU enumeration and scanning electron microscopy.

Integrated Stress Response and Immunopathogenic Assays

ISR activation was quantified by western blotting for phosphorylated eIF2α (Ser51), total eIF2α, PERK,

GCN2, PKR, HRI, ATF4, and CHOP, normalized to GAPDH and β-actin. qRT-PCR assessed

transcriptional dynamics of EIF2AK3, ATF4, DDIT3, GADD34, and inflammatory mediators (IL1B,

IL6, CXCL8, CCL20, TNF). Barrier integrity was evaluated using transepithelial electrical resistance

(TEER) measurements (EVOM2), FITC-dextran (4 kDa) paracellular flux assays, and confocal

immunostaining of ZO-1, claudin-4, and occludin. Inflammasome activation was assessed via ASC speck

formation (immunofluorescence), caspase-1 p20 activity (FLICA assay), and IL-1β/IL-18 secretion

(Luminex multiplex). Cytokine profiles were analyzed using a 38-plex human inflammation panel.

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Pharmacological and Genetic Modulation

To establish causal relationships, ISR signaling was pharmacologically modulated using ISRIB (100

nM), PERK inhibitor GSK2606414 (250 nM), GCN2 inhibitor A-92 (5 μM), and salubrinal (5 μM).

Genetic perturbation employed lentiviral CRISPR-Cas9 knockout or siRNA transfection targeting

EIF2AK3, EIF2AK4 (GCN2), ATF4, and DDIT3. Non-targeting controls and scrambled siRNAs were

utilized. Rescue experiments co-administered ISR inhibitors following bacterial challenge to differentiate

adaptive from maladaptive ISR phases. Transfection efficiency (>80%) and off-target effects were

validated via qRT-PCR and Sanger sequencing.

Multi-Omics Profiling and Spatial Transcriptomics

Single-cell RNA sequencing (scRNA-seq) was performed using the 10x Genomics Chromium Next

GEM system (v3.1) on dissociated clinical biopsies and in vitro cultures, targeting 10,000 cells per

sample. Spatial transcriptomics utilized 10x Genomics Visium HD slides on fresh-frozen gingival

sections, capturing epithelial compartment-specific expression gradients. Targeted phosphoproteomics

(Olink Explore 3072) quantified ISR and inflammatory signaling nodes. Bulk RNA-seq libraries were

prepared using Illumina TruSeq Stranded mRNA kits. All omics workflows included spike-in controls,

batch randomization, and technical replicates.

In Vivo Validation of ISR-Mediated Periodontal Immunopathology

C57BL/6J mice (8-week-old, male) underwent silk ligature placement around maxillary second molars

for 10 days to induce experimental periodontitis. Local subgingival delivery of ISRIB (10 mg/kg in

Pluronic F127 gel) or vehicle was administered on days 3, 5, and 7. Alveolar bone loss was quantified

via micro-CT (μXCT 35, voxel size 10 μm) and histomorphometry (cementoenamel junction–alveolar

bone crest distance). Gingival tissues were processed for IHC (p-eIF2α, ATF4, CHOP, MPO), flow

cytometry (CD45⁺/Ly6G⁺/CD11b⁺/CD3⁺ subsets), and cytokine quantification. Microbiome

composition was assessed via 16S rRNA amplicon sequencing of ligature-adjacent plaque.

Data Processing, Bioinformatics, and Statistical Analysis

scRNA-seq data were processed using Cell Ranger (v7.2) and analyzed in R (v4.3.2) with Seurat

(v4.3.0). Quality control filters excluded cells with <200 genes, >20% mitochondrial reads, or doublet

scores >0.25 (DoubletFinder). Dimensionality reduction employed PCA, UMAP, and Louvain

clustering. Spatial transcriptomics data were aligned using SpaceRanger (v2.0) and analyzed with Squidpy

and Giotto. Differential expression utilized DESeq2 and edgeR with Benjamini–Hochberg false

discovery rate (FDR < 0.05) correction. Pathway enrichment employed GSEA, Enrichr, and IPA.

Phosphoproteomic data were normalized using variance-stabilizing transformation and subjected to

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network analysis via Cytoscape (STRING plugin). Statistical analyses were performed in GraphPad

Prism 10.0 and R. Normality was assessed via Shapiro–Wilk tests. Group comparisons employed two-

way ANOVA with Tukey’s post hoc test for multi-factor designs, or Kruskal–Wallis with Dunn’s

correction for non-parametric data. Longitudinal and repeated-measures data were analyzed using linear

mixed-effects models (lme4 package) with random intercepts for donor/mouse. Correlation analyses

applied Spearman’s rank test with FDR adjustment.

Reproducibility, Blinding, and Quality Control

All experiments were conducted in ≥3 independent biological replicates with technical triplicates.

Investigators were blinded to group allocation during data acquisition and analysis. Reagent lots, passage

numbers, and culture conditions were standardized and documented. Negative and positive controls were

included in every assay batch. Inter-assay variability was monitored via coefficient of variation (<15%

for ELISA/Luminex, <10% for qRT-PCR). Batch effects in omics data were corrected using ComBat-

seq and Harmony integration. Raw and processed datasets, along with metadata, will be deposited in

GEO (scRNA-seq/spatial transcriptomics) and PRIDE (phosphoproteomics) prior to publication.

Results and Analysis

Clinical Specimen Characterization Reveals Elevated ISR Activation in Periodontitis

Table:01 Human Gingival Biopsy Analysis: ISR Marker Expression in Periodontitis vs. Health Patient Cohort Characteristics

Periodontitis Group

(Stage III/IV, Grade C; n =

12)

Healthy Controls

Parameter

Statistical Comparison

(n = 12)

Mean Age (years)

48.3 ± 9.2

7:5

6.8 ± 1.4

45.7 ± 10.1

6:6

2.1 ± 0.4

p = 0.52 (ns)

Fisher's exact p = 1.0

p < 0.001

Sex Distribution (F:M)

Mean Probing Depth (mm)

Mean Clinical Attachment Loss

(mm)

Bleeding on Probing (%)

Smoking Status

5.9 ± 1.7

78.4 ± 12.3

4/5/3

0.3 ± 0.2

8.2 ± 3.1

3/4/5

p < 0.001

p = 0.68 (ns)

(Current/Former/Never)

Table: 02 Immunohistochemical Quantification of ISR Markers in Epithelial Compartments

Healthy Controls

(Mean ± SD)

Periodontitis

(Mean ± SD)

Statistical

Significance

Marker / Compartment

Fold Change

Effect Size

p-eIF2α (Ser51)

Sulcular Epithelium

Junctional Epithelium

Nuclear ATF4

12.4 ± 3.1 H-score

10.8 ± 2.7 H-score

47.2 ± 8.9 H-score

45.3 ± 9.4 H-score

3.8×

4.2×

Cohen's d = 2.1

Cohen's d = 2.4

p < 0.001

Sulcular Epithelium

Junctional Epithelium

Cytoplasmic CHOP

Sulcular Epithelium

Junctional Epithelium

8.2 ± 2.4 H-score

7.5 ± 2.1 H-score

31.6 ± 7.1 H-score

29.8 ± 6.8 H-score

3.9×

4.0×

Cohen's d = 1.9

Cohen's d = 2.0

p < 0.01

6.9 ± 1.9 H-score

6.1 ± 1.7 H-score

26.4 ± 5.3 H-score

24.9 ± 4.9 H-score

3.8×

4.1×

Cohen's d = 1.8

Cohen's d = 1.9

p < 0.01

Gingival biopsies from patients with stage III/IV, grade C periodontitis (n = 12) exhibited significantly

elevated expression of integrated stress response (ISR) markers compared to periodontally healthy

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controls (n = 12). Immunohistochemical quantification demonstrated a 3.8-fold increase in

phosphorylated eIF2α (Ser51) in sulcular epithelium (p < 0.001, Cohen's d = 2.1) and a 4.2-fold

increase in junctional epithelium (p < 0.001, Cohen's d = 2.4) of diseased tissues (Figure 1A–C).

Concurrently, nuclear ATF4 and cytoplasmic CHOP expression were significantly upregulated in both

epithelial compartments (p < 0.01 for all comparisons), correlating strongly with clinical attachment loss

(r = 0.78, p = 0.003) and probing depth (r = 0.71, p = 0.009) (Figure 1D). Bulk RNA-seq analysis

confirmed transcriptional upregulation of EIF2AK3 (PERK; log₂FC = 2.3, FDR = 0.004), ATF4

(log₂FC = 1.9, FDR = 0.012), and DDIT3 (CHOP; log₂FC = 2.7, FDR = 0.002) in periodontitis

specimens, with pathway enrichment revealing significant activation of the unfolded protein response and

inflammatory signaling networks (GSEA NES = 2.1, FDR < 0.001). These findings establish that ISR

activation is a prominent feature of the periodontal epithelium in human disease and correlates with

clinical severity.

Primary Keratinocyte Isolation Confirms Compartment-Specific ISR Dynamics

Fluorescence-activated cell sorting successfully isolated highly pure populations of junctional

keratinocytes

(ITGA6⁺/ITGB4⁺/EPCAM⁺;

>94%

purity)

and

sulcular

keratinocytes

(KRT13⁺/KRT4⁺/EPCAM⁺; >91% purity) from healthy and diseased gingival tissues (Figure 2A).

Baseline scRNA-seq profiling revealed distinct transcriptional signatures between compartments:

junctional keratinocytes exhibited elevated expression of attachment-related genes (ITGA6, ITGB4,

COL17A1), while sulcular keratinocytes showed enrichment for barrier-regulatory transcripts (CLDN4,

OCLN, TJP1). Upon exposure to a dysbiotic microbial consortium (P. gingivalis, T. forsythia, F.

nucleatum), both populations activated ISR signaling, but with compartment-specific kinetics: junctional

keratinocytes exhibited earlier PERK/eIF2α phosphorylation (peak at 6 h; p < 0.001), whereas sulcular

keratinocytes showed delayed but sustained GCN2 activation (peak at 24 h; p < 0.001) (Figure 2C–E).

These data indicate that epithelial subpopulations deploy distinct ISR kinase strategies in response to

identical microbial challenges, potentially reflecting their specialized anatomical functions.

Dysbiotic Microbiota Selectively Activate ISR Kinases via Distinct Virulence Mechanisms

Controlled exposure experiments revealed that individual pathobionts engage specific ISR upstream

kinases through defined virulence mechanisms. P. gingivalis gingipains induced robust PERK

phosphorylation and eIF2α activation (p < 0.001), an effect abrogated by the gingipain inhibitor Z-

Phe-Ala-diazomethylketone (p = 0.002). In contrast, F. nucleatum outer membrane vesicles triggered

PKR activation via double-stranded RNA delivery (p < 0.001), while amino acid deprivation mimicking

the inflamed sulcus microenvironment selectively activated GCN2 (p < 0.001). Health-associated

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commensals (S. sanguinis, V. parvula) induced transient, low-amplitude ISR activation that resolved

within 24 h, whereas dysbiotic consortia sustained eIF2α phosphorylation beyond 72 h (p < 0.001 for

time × condition interaction). Phosphoproteomic profiling identified 47 ISR-associated phosphosites

significantly enriched during dysbiotic exposure, including novel PERK substrates involved in

mitochondrial quality control. These results demonstrate that dysbiotic microbes exploit multiple stress-

sensing pathways to establish chronic ISR activation in gingival keratinocytes.

ISR Activation Amplifies Pro-Inflammatory Signaling and Compromises Epithelial Barrier Integrity

Table: 03

Key Molecular /

Functional Outcome

Direct occupancy at

IL1B, IL6, CXCL8,

CCL20 promoters

Statistical

Significance

Experimental Approach

Target / Intervention

Quantitative Result

ATF4 binding to

cytokine gene

promoters

Binding intensity ↔

transcript abundance: r =

0.82

ATF4 ChIP-seq

p < 0.001

IL-1β/CXCL8:

p < 0.001; IL-

6: p = 0.003

p < 0.01 for all

cytokines

Pharmacological ISR

Inhibition

IL-1β: −68%; IL-6:

−54%; CXCL8: −61%

↓ Pro-inflammatory

cytokine secretion

ISRIB (100 nM)

Pharmacological ISR

Inhibition

GSK2606414 (PERK

antagonist, 250 nM)

ISRIB or

GSK2606414

treatment

Comparable attenuation

to ISRIB

↓ Pro-inflammatory

cytokine secretion

72% reduction vs.

dysbiosis-only control

↓ NF-κB nuclear

translocation

p < 0.001

NF-κB Signaling Analysis

Dysbiosis: p <

0.001; ISRIB

rescue: p =

0.004

Dysbiosis: −45%

TEER; ISRIB co-

treatment: full recovery

Dysbiotic exposure ±

ISRIB co-treatment

TEER decline reversed

by ISR inhibition

Barrier Function (TEER)

CHOP-dependent

downregulation of

junctional proteins

Claudin-4 & occludin

junctional intensity

restored

2.8-fold ↑ flux vs.

homeostatic control;

ISRIB normalizes

Tight Junction Protein

Localization

CHOP knockdown /

ISR inhibition

p < 0.001

FITC-dextran flux

across keratinocyte

monolayers

Paracellular Permeability

Assay

ISR activation increases

epithelial permeability

Sustained ISR activation directly enhanced immunopathogenic outputs in primary keratinocytes. ATF4

chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed direct binding to promoter

regions of IL1B, IL6, CXCL8, and CCL20, with binding intensity correlating with transcript abundance

(r = 0.82, p < 0.001). Pharmacological ISR inhibition with ISRIB (100 nM) or PERK-selective

antagonist GSK2606414 (250 nM) significantly attenuated cytokine secretion (IL-1β: −68%, p <

0.001; IL-6: −54%, p = 0.003; CXCL8: −61%, p < 0.001) and reduced NF-κB nuclear translocation

by 72% (p < 0.001). Concurrently, ISR activation compromised barrier function: TEER values declined

by 45% following dysbiotic exposure (p < 0.001), an effect reversed by ISRIB co-treatment (p = 0.004).

Confocal microscopy revealed CHOP-dependent downregulation of claudin-4 and occludin at cell–cell

junctions (p < 0.001), while FITC-dextran flux assays confirmed increased paracellular permeability (p

< 0.001). These findings establish a causal link between ISR signaling, amplified inflammation, and

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epithelial barrier failure.

Pharmacological and Genetic ISR Modulation Attenuates Immunopathogenic Outputs

Table: 04

Intervention

CRISPR-Cas9

Knockout

Molecular Target

Primary Effect

↓ Pro-inflammatory

cytokine secretion

↓ Pro-inflammatory

cytokine secretion

↑ Epithelial barrier

preservation

Key Readouts

Statistical Significance

EIF2AK3 (PERK)

p < 0.001

IL-1β, CXCL8

CRISPR-Cas9

Knockout

ATF4

p < 0.001

p = 0.002

IL-1β, CXCL8

CRISPR-Cas9

Knockout

DDIT3 (CHOP)

TEER recovery

Salubrinal (blocks

eIF2α

↑ Inflammatory &

barrier-disruptive

responses

Pharmacological

Inhibition

Exacerbated cytokine

release, TEER decline

p = 0.008

dephosphorylation)

Integrated Stress

Response (0–6 h post-

exposure)

Maintained TEER,

normal epithelial

morphology

ISRIB Administration

(Early)

Prevented barrier

dysfunction

Significant protection

Not significant

Integrated Stress

Response (24–48 h

post-exposure)

ISRIB Administration

(Delayed)

Failed to restore

homeostasis

No TEER recovery;

persistent dysfunction

To validate the functional contribution of specific ISR components, we employed targeted genetic and

pharmacological interventions. CRISPR-Cas9 knockout of EIF2AK3 (PERK) or ATF4 in junctional

keratinocytes significantly reduced dysbiosis-induced IL-1β and CXCL8 secretion (p < 0.001 for both),

whereas DDIT3 (CHOP) knockout preserved barrier integrity without affecting cytokine output (p =

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0.002 for TEER recovery). Salubrinal-mediated eIF2α dephosphorylation inhibition exacerbated

inflammatory responses (p = 0.008), confirming that adaptive ISR resolution is essential for limiting

immunopathology. Time-course rescue experiments demonstrated that early ISRIB administration (0–6

h post-exposure) prevented barrier dysfunction, whereas delayed treatment (24–48 h) failed to restore

homeostasis, defining a critical therapeutic window for ISR modulation. These data underscore the

phase-dependent consequences of ISR signaling and support targeted, temporally precise therapeutic

strategies.

Multi-Omics Integration Reveals ISR-Centered Regulatory Networks in Periodontal Epithelium

Integrated analysis of scRNA-seq, spatial transcriptomics, and phosphoproteomics identified a core ISR-

centered regulatory network governing epithelial immunopathology. Weighted gene co-expression

network analysis (WGCNA) clustered 1,247 dysbiosis-responsive genes into 18 modules, with the

"turquoise" module (enriched for ISR, NF-κB, and tight junction genes) showing the strongest

correlation with disease severity (r = 0.89, p < 0.001). Spatial transcriptomics mapped ATF4 and

CHOP expression gradients extending from the junctional epithelium into the underlying connective

tissue, colocalizing with infiltrating CD68⁺ macrophages and MPO⁺ neutrophils. Phosphoproteomic

network analysis positioned PERK-mediated eIF2α phosphorylation as a central hub connecting

microbial sensing to downstream inflammatory and barrier-disruptive effectors (betweenness centrality

= 0.34, top 5% of network). Machine learning classification (random forest) using ISR marker

expression accurately distinguished periodontitis from health (AUC = 0.94, 95% CI [0.88, 0.99]),

highlighting the diagnostic potential of epithelial ISR signatures.

In Vivo Validation Confirms ISR Inhibition Attenuates Periodontal Immunopathology

Table: 05

Outcome Measure

ISRIB-Treated Group

Vehicle-Treated Control

Statistical Comparison

Alveolar Bone Loss

p < 0.001; effect size =

3.2

Mean CEJ–ABC Distance

142 ± 18 μm

218 ± 24 μm

Micro-CT Volumetric Analysis

Bone Volume/Total Volume

(BV/TV)

38.2% ± 4.1%

Preserved

24.7% ± 3.8%

Compromised

p < 0.001

Trabecular Architecture

Histological Assessment

Epithelial Ulceration

—

Reduced

Prominent

Qualitative improvement

p < 0.001

−64% reduction vs.

Baseline infiltration

Neutrophil Infiltration (MPO⁺ cells)

control

Significantly lower

Elevated

p < 0.01

Gingival IL-1β Levels

Gingival TNF-α Levels

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Outcome Measure

ISRIB-Treated Group

Vehicle-Treated Control

Statistical Comparison

Microbial Community Analysis (16S

rRNA)

In a murine ligature-induced periodontitis model, local subgingival delivery of ISRIB (10 mg/kg)

significantly reduced alveolar bone loss compared to vehicle-treated controls (mean CEJ–ABC distance:

142 ± 18 μm vs. 218 ± 24 μm; p < 0.001, effect size = 3.2). Micro-CT volumetric analysis confirmed

preservation of trabecular bone architecture in ISRIB-treated mice (bone volume/total volume: 38.2%

± 4.1% vs. 24.7% ± 3.8%; p < 0.001). Histological assessment revealed reduced epithelial ulceration,

decreased neutrophil infiltration (MPO⁺ cells: −64%, p < 0.001), and lower gingival IL-1β and TNF-

α levels (p < 0.01 for both) in the ISRIB group . 16S rRNA sequencing of ligature-adjacent plaque

showed no significant differences in microbial community composition between treatment groups

(PERMANOVA p = 0.34), indicating that ISRIB's protective effects were mediated through host

modulation rather than antimicrobial activity. These in vivo findings validate the translational relevance

of targeting epithelial ISR signaling to mitigate periodontal tissue destruction.

Spatial Transcriptomics Maps ISR Activity Gradients in Human Periodontal Tissues

High-resolution spatial transcriptomics of human gingival biopsies revealed compartment-specific ISR

activity patterns that align with disease progression. In healthy tissues, low-level ATF4 expression was

confined to basal keratinocyte layers, whereas periodontitis specimens exhibited strong ATF4 and CHOP

signals extending through suprabasal sulcular and junctional epithelium. Cell-type deconvolution

identified

a

disease-associated

keratinocyte

subpopulation

(characterized

by

ATF4⁺/CHOP⁺/CXCL8⁺/MMP9⁺ signature) that was significantly enriched in periodontitis tissues

(12.3% ± 2.1% vs. 1.8% ± 0.7% in health; p < 0.001) and spatially colocalized with regions of

epithelial erosion and immune cell infiltration. Pseudotemporal ordering analysis suggested that ISR-

high keratinocytes represent a transitional state preceding barrier failure and inflammatory amplification.

These spatially resolved data provide unprecedented insight into the microanatomical distribution of

ISR-driven immunopathology and identify potential targets for precision epithelial therapies.

Conclusion

This study establishes that dysbiotic subgingival microbiota chronically activate the integrated stress

response (ISR) in sulcular and junctional keratinocytes, converting an evolutionarily conserved

cytoprotective pathway into a central amplifier of periodontal immunopathology. Sustained eIF2α

phosphorylation and downstream ATF4/CHOP signaling drive excessive pro-inflammatory cytokine

release, prime NLRP3 inflammasome activation, and disrupt tight junction integrity, thereby accelerating

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epithelial barrier failure, connective tissue degradation, and alveolar bone resorption. Targeted

pharmacological and genetic attenuation of ISR signaling effectively mitigates these destructive processes

without perturbing microbial ecology, demonstrating that host-directed epithelial modulation represents

a viable adjunctive strategy for periodontal therapy. Collectively, these findings reposition the

keratinocyte ISR axis as a critical molecular nexus linking microbial dysbiosis to tissue-destructive

inflammation and provide a mechanistic foundation for precision interventions in periodontal disease.

Future research should prioritize longitudinal, single-cell and spatially resolved profiling to define the

precise temporal threshold at which adaptive ISR signaling transitions to maladaptive immunopathology

in distinct keratinocyte subpopulations. Development of locally delivered, kinase-selective ISR

modulators (e.g., PERK/GCN2 inhibitors, eIF2α phosphatase enhancers) requires rigorous

pharmacokinetic optimization and phase I/II clinical evaluation to ensure epithelial safety and

therapeutic efficacy. Integrating multi-omics platforms with metabolomic and microbiome sequencing

will further clarify how epithelial stress states shape subgingival ecological resilience and immune cell

recruitment. Additionally, validation of ISR-associated epithelial signatures in gingival crevicular fluid or

saliva could yield noninvasive biomarkers for early disease stratification and treatment response

monitoring. Ultimately, combinatorial regimens that simultaneously restore epithelial barrier function,

recalibrate maladaptive inflammatory signaling, and promote microbiome stability will define the next

generation of host-directed periodontal therapeutics.

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GRJNST, Volume: 04 - Issue 2 (2026) / ISSN P: 2790-7643

Article ID: 2077

https://doi.org/10.53762/grjnst.04.02.28