Methamphetamine use and HSV-1 coinfection synergistically alter IFNα, IFNγ, and IL-29 expression in gingival crevicular fluid: implications for gingivitis pathogenesis

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Introduction

Methamphetamine is a potent central nervous system stimulant chemically classified as a phenethylamine (C10H15N). Chronic use of this substance is associated with severe health consequences, including cardiovascular disease, neurological dysfunction, immune suppression, and significant oral health deterioration [2, 8]. A hallmark condition of long-term methamphetamine abuse is “meth mouth”, characterized by rampant dental decay, xerostomia, periodontal disease, and tooth loss [17]. This condition arises from reduced saliva production, poor oral hygiene, high sugar consumption, and behaviors like bruxism, all of which collectively promote bacterial proliferation and gingival inflammation.

Drug addiction, including methamphetamine use, is a chronic relapsing disorder marked by compulsive drug-seeking behavior and persistent use despite negative consequences [34]. It affects not only the individual but also their family and community. The systemic effects of addiction are wide-ranging, leading to physical and psychological impairments, notably in brain function and immune regulation [16, 26]. Methamphetamine has been shown to suppress immune responses, increasing vulnerability to infections, including viral pathogens like Herpes Simplex Virus type 1 (HSV-1) [11, 34].

HSV-1, a member of the Herpesviridae family, typically establishes latency in the trigeminal ganglia and periodically reactivates. It is primarily associated with oral infections and can exacerbate periodontal conditions such as gingivitis, especially when coexisting with bacterial plaque accumulation [9]. Clinical signs may intensify in co-infected individuals, presenting with increased gingival erythema, edema, ulceration, and bleeding [11, 28]. The combined impact of meth use and HSV-1 infection may synergistically impair oral immunity, contributing to more severe periodontal manifestations.

Gingivitis, the earliest form of periodontal disease, is a reversible inflammatory condition primarily induced by dental plaque. It is histologically characterized by collagen degradation, inflammatory cell infiltration, and vasodilation [11, 14, 20, 36]. While generally non-destructive in its early stages, untreated gingivitis can progress to periodontitis and systemic inflammatory responses. Bacteria such as Streptococcus mutans and Porphyromonas gingivalis play central roles in its pathogenesis [1, 21, 37, 38, 43].

The immune response to both viral and bacterial infections involves the production of cytokines and chemokines, including interferons (IFNs) and interleukin-29 (IL-29), which modulate inflammation and antiviral defense. Type I (IFNα) and Type II (IFNγ) interferons are pivotal in activating macrophages, NK cells, and T-cells, enhancing antigen presentation and immune surveillance [11, 37, 38]. Though IFNs have clinical utility in cancer immunotherapy, their presence in the oral cavity reflects immune activation against infection more so than oncologic relevance in this context.

IL-29 (a Type III interferon) has emerged as an important immunomodulator in mucosal tissues. It is elevated in gingival crevicular fluid during periodontal inflammation and may be induced by HSV-1 infection. IL-29 has been shown to enhance antiviral responses and is positively correlated with clinical markers such as pocket depth and attachment loss, indicating its involvement in disease progression [11, 37].

This study aimed to evaluate and compare the levels of IFNα, IFNγ, and IL-29 in gingival crevicular fluid among methamphetamine users — with and without HSV-1 infection — alongside patients with plaque-induced gingivitis and healthy individuals.

Materials and methods

Study design. This case-control study was conducted between November 2024 and May 2025, involving 88 male participants aged 20–35 years, recruited from dental clinics and specialized healthcare centers. Participants were stratified into four distinct groups: Healthy Controls (n = 25) with no history of systemic or periodontal disease; Methamphetamine Group (n = 22) comprising confirmed meth users without evidence of HSV infection; Meth with HSV Group (n = 21), including meth users with clinical or serological confirmation of HSV-1 infection; and Gingivitis Group (n = 20), consisting of non-drug users diagnosed with gingivitis and negative for HSV. The sample size was determined using the EPITOOLS online calculator, based on a 95% confidence level and an alpha of 0.05.

Ethical approval. All procedures adhered to ethical standards approved by the Scientific Committee of the Basic Science Department, College of Dentistry, University of Baghdad (Ref: 959, Project No: 959824, Date: 24.10.2024). Written informed consent was obtained from all participants.

Included participants

• Males aged 20–35
• Systemically healthy and consistent with group classification
• Free from immunosuppressive therapy

Excluded

• History of systemic illness or autoimmune disorders
• Use of medications affecting immune function
• Substance abuse other than Meth
• Diabetes or other unrelated chronic conditions

Clinical periodontal assessment

A trained periodontist examined all teeth using a standardized protocol.

• Bleeding on Probing (BOP%): Assessed at six sites per tooth using gentle probing.
• Plaque Index (PLI): Quantified the extent and thickness of plaque based on standard scoring criteria.

GCF sampling and calibration. Gingival crevicular fluid (GCF) was collected using PerioPaper strips from four sites on upper first premolars (mesiobuccal, distobuccal, mesiopalatal, distopalatal) between 9:00 AM and 2:30 PM. Strips contaminated with blood/saliva were discarded. GCF volume was quantified using a Periotron 8010 (OraFlow Inc., USA), calibrated via serial dilutions (0.25–1.25 μL distilled water) to generate a standard curve. Paper strips were immersed in 0.5 mL sterile PBS, centrifuged at 3000 rpm for 10 min, and stored at –20°C until ELISA testing.

Concentration (pg/μL) = ELISA result (pg/mL) × 0.2/GCF volume (μL)

ELISA analysis of cytokines. ELISA kits sourced from Biotech (Shanghai) were utilized to quantify IFNα (Cat. CK-bio-12065), IFNγ (Cat. CK-bio-12068), and IL-29 (Cat. CK-bio-12121), each based on the double-antibody sandwich principle. In this method, samples and standards were added to wells pre-coated with specific antibodies, followed by the addition of HRP-conjugate reagent to form immune complexes. After incubation and thorough washing to remove unbound components, substrate solutions were introduced to develop a colorimetric reaction, which was stopped with an acid solution. The optical density (OD) was measured at 450 nm using a microplate reader. Cytokine concentrations were determined by comparing sample OD values to standard curves generated through regression analysis, with final results adjusted according to the appropriate dilution factors.

Statistical analysis. Data were analyzed using SPSS version 26. Normality of distribution was assessed using the Shapiro–Wilk test. Descriptive statistics were reported as means±standard deviation for normally distributed (parametric) variables. For inferential analysis, one-way ANOVA followed by LSD post hoc tests was applied to compare means among groups. Pearson correlation coefficients were used to examine relationships between cytokine levels parameters. Chi-square tests were applied where appropriate. A p-value < 0.05 was considered statistically significant.

Results

Table 1 illustrates the distribution of age among the four study groups: healthy, methamphetamine users (Meth), methamphetamine users with HSV infection (Meth with HSV), and gingivitis patients. The mean age was highest in the Meth group (26.50±4.52 years), followed by Meth with HSV (25.14±4.39 years), while the lowest mean age was observed in the Gingivitis group (23.45±1.67 years). The healthy group had a mean age of 23.76±2.19 years. A one-way ANOVA analysis revealed a statistically significant difference in age distribution among the groups (F = 3.672, p = 0.015).

 

Table 1. Age (year) distribution among groups

Study groups	N	Mean	Std. Deviation	Std. Error of Mean	F	p-value
Healthy	25	23.76	2.185	0.437	3.672	0.015
Meth	22	26.50	4.522	0.964
Meth with HSV	21	25.14	4.385	0.957
Gingivitis	20	23.45	1.669	0.373
Total	88	24.70	3.572	0.381

 

The results showed noticeable variation in IFNα levels across the study groups was shown in Table 2. The healthy group exhibited the highest mean concentration (193.97±31.61 pg/mL), followed by the Meth with HSV group (182.54±13.17 pg/mL) and the Meth group (165.91±16.80 pg/mL), while the lowest levels were observed in the Gingivitis group (132.63±44.20 pg/mL). One-way ANOVA analysis indicated a statistically significant difference among the groups (p = 0.030).

 

Table 2. Interferon alpha (IFNα) levels in all groups — one-way ANOVA analysis

	Mean	Std. Deviation	Std. Error	F	p-value
Healthy	193.97	31.61	6.32	3.130	0.030
Meth	165.91	16.80	3.58
Meth with HSV	182.54	13.17	2.86
Gingivitis	132.63	44.20	9.88

 

By using post hoc Games–Howell test, the healthy group had significantly higher IFNα levels than both the Meth group (p = 0.002) and the Gingivitis group (p = 0.000). Additionally, the Meth group showed a significantly higher level than the Gingivitis group (p = 0.020). However, no significant differences were detected between the Meth and Meth with HSV groups, nor between the Meth with HSV and Gingivitis groups, as shown in Table 3.

 

Table 3. Post hoc Games–Howell multiple comparisons of IFNα levels between study groups

Study groups	Mean Difference	p-value	Sig.
Healthy vs Meth	28.07*	0.002	Sig.
Healthy vs Meth with HSV	11.44	0.979	Non-Sig.
Healthy vs Gingivitis	61.34*	0.000	Sig.
Meth vs Meth with HSV	–16.63	0.938	Non-Sig.
Meth vs Gingivitis	33.28*	0.020	Sig.
Meth with HSV vs Gingivitis	49.91	0.372	Non-Sig.

 

The results of IFNγ levels comparisons across the study groups was observed in Table 4.

 

Table 4. Interferon gamma (IFNγ) levels in all groups — one-way ANOVA analysis

	Mean	Std. Deviation	Std. Error	F	p-value
Healthy	139.22	24.42	4.88	145.168	0.0001
Meth	108.55	13.32	2.84
Meth with HSV	75.58	8.99	1.96
Gingivitis	45.87	8.58	1.92

 

The healthy group had the highest mean IFNγ level (139.22±24.42 pg/mL), followed by the Meth group (108.55±13.32 pg/mL), the Meth with HSV group (75.58±8.99 pg/mL), and the lowest level was recorded in the Gingivitis group (45.87±8.58 pg/mL). One-way ANOVA revealed a highly significant difference among the groups (p < 0.0001).

The healthy group had significantly higher IFNγ levels than the Meth (p = 0.000), Meth with HSV (p = 0.000), and Gingivitis groups (p = 0.000). Additionally, the Meth group showed significantly elevated IFNγ levels compared to the Meth with HSV (p = 0.000) and Gingivitis groups (p = 0.000), while the Meth with HSV group also had significantly higher levels than the Gingivitis group (p = 0.000), as shown in Table 5.

 

Table 5. Post hoc Games–Howell multiple comparisons of IFNγ levels between study groups

Study_groups	Mean Difference	p-value	Sig.
Healthy vs Meth	30.67*	0.000	Sig.
Healthy vs Meth with HSV	63.64*	0.000	Sig.
Healthy vs Gingivitis	93.35*	0.000	Sig.
Meth vs Meth with HSV	32.97*	0.000	Sig.
Meth vs Gingivitis	62.68*	0.000	Sig.
Meth with HSV vs Gingivitis	29.71*	0.000	Sig.

 

The Gingivitis group exhibited the highest mean IL-29 level (257.14±4.53 pg/mL), followed by the Meth with HSV group (241.45±4.75 pg/mL), and the Meth group (226.43±4.84 pg/mL). In contrast, the healthy group showed the lowest IL-29 concentration (205.75±21.59 pg/mL). One-way ANOVA revealed a statistically significant difference among the groups (p = 0.000), as demonstrated in Table 6.

 

Table 6. Interleukin-29 (IL-29) levels in all groups — one-way ANOVA analysis

	Mean	Std. Deviation	Std. Error	F	p-value
Healthy	205.75	21.59	4.32	72.358	0.000
Meth	226.43	4.84	1.03
Meth with HSV	241.45	4.75	1.04
Gingivitis	257.14	4.53	1.01

 

The Gingivitis group showed significantly higher IL-29 levels than the Meth with HSV group (p = 0.000), the Meth group (p = 0.000), and the healthy group (p = 0.000). Likewise, the Meth with HSV group exhibited elevated IL-29 levels compared to the Meth group (p = 0.000) and the healthy group (p = 0.000). The Meth group also demonstrated significantly higher IL-29 levels than the healthy group (p = 0.000), as illustrated in Table 7.

 

Table 7. Post hoc Games–Howell multiple comparisons of IL-29 levels between study groups

Study groups	Mean Difference	p-value	Sig.
Healthy vs Meth	–20.68*	0.000	Sig.
Healthy vs Meth with HSV	–35.70*	0.000	Sig.
Healthy vs Gingivitis	–51.39*	0.000	Sig.
Meth vs Meth with HSV	–15.024*	0.000	Sig.
Meth vs Gingivitis	–30.71*	0.000	Sig.
Meth with HSV vs Gingivitis	–15.69*	0.000	Sig.

 

Discussion

The present study examined the age distribution across four distinct groups: healthy individuals, methamphetamine users, meth users co-infected with HSV, and patients with gingivitis. The analysis revealed that methamphetamine users were generally older than participants in the other groups. Those with both meth use and HSV infection also tended to be older, though slightly younger than meth users without HSV. In contrast, gingivitis patients and healthy controls were the youngest among all groups. Statistical testing confirmed that the variation in age across these groups was significant.

Methamphetamine users tend to be older than those in healthy and gingivitis-only groups. This is consistent with Shetty et al. (2015) [31] reported that the majority of methamphetamine users are between 18 and 34 years old, with oral health consequences becoming more pronounced with longer duration of use.

Concerning Gingivitis with age groups, in the current study it is more prevalent among younger individuals, particularly adolescents and young adults, these may be due to factors such as poor oral hygiene, hormonal changes, and lifestyle habits. Furthermore, Muqarab et al. (2024) [22] and Alwan (2024) [3] found that the highest prevalence of gingivitis is in the 15–25 year age group, with the frequency decreasing as age increases, while periodontitis becomes more common in older populations.

Regarding Methamphetamine, HSV infection, and age in this study, Methamphetamine users with HSV infection (meth with HSV) had a mean age between that of meth users and the other groups. This may reflect the cumulative risk behaviors associated with both substance use and viral infection, which tend to manifest in early adulthood and may be linked to longer exposure periods [40].

These findings suggest a trend where older individuals are more likely to be methamphetamine users. However, those in the slightly younger age range appeared more frequently in the group with both meth use and HSV infection. This could point to increased vulnerability to viral infections like HSV among younger meth users. Moreover, the youngest age group showed a higher prevalence of gingivitis, possibly reflecting early-stage gum disease that develops before more severe complications arise.

The investigation of IFNα (interferon-alpha) levels across four groups—healthy controls, methamphetamine users (meth), meth users with HSV infection (meth + HSV), and gingivitis patients—revealed a clear hierarchy. Moreover, the healthy group showed the highest IFNα concentration, followed by the meth + HSV and meth groups, while the gingivitis group had the lowest levels.

These patterns suggest that methamphetamine use impairs the ability of immune cells, including plasmacytoid dendritic cells and antigen-presenting cells, to produce IFNα—even in the presence of viral infection. Indeed, chronic meth exposure has been shown to suppress endogenous IFNα production in macrophages and antigen-presenting cells, weakening critical innate antiviral defenses [18]. The significant drop in IFNα among meth users compared to healthy individuals reflects this immunosuppressive effect, a deficit not substantially worsened by HSV co-infection, which aligns with the lack of significant difference between meth and meth + HSV groups.

Meanwhile, the lowest IFNα levels in the gingivitis group likely reflect the nature of localized, bacterial-driven inflammation. Furthermore, prior studies report that while total IFNα amounts may not differ much between healthy and diseased periodontal sites, the concentration of IFNα is significantly reduced at inflamed sites — possibly due to increased fluid volume diluting cytokines or impaired local production.

Furthermore, it appears that methamphetamine use significantly undermines innate immune signaling through IFNα suppression, even more than local bacterial inflammation associated with gingivitis. Meanwhile, healthy individuals maintain robust IFNα levels, which likely contribute to more effective antiviral surveillance. Moreover, HSV co-infection did not restore IFNα levels among meth users, suggesting that meth’s inhibitory impact on immune cell function outweighs the inflammatory stimulus provided by viral presence.

According to Mathur et al. (1996) [19] and de Menezes et al. (2018) [7] IFNα is crucial for the early immune response, limiting pathogen replication and supporting tissue homeostasis.

Salamanca et al. (2014) [29], Passaro et al. (2015) [24] and Borgmann and Ghorpade (2015) [5] reported that Methamphetamine is known to suppress IFNα production and impair immune responses, making users more susceptible to infections and inflammatory complications, this supportd the current finding for IFNα levels were reduced in meth users compared to healthy controls.

Mathur et al. (1996) [14] and shown that IFNα concentrations are significantly lower in diseased gingival sites compared to healthy sites, even when the total amount of IFNα remains similar. This is attributed to increased crevicular fluid volume in disease, which dilutes cytokine concentrations.

The study revealed that healthy individuals had the highest IFNγ levels, followed by meth users, then meth + HSV users, with gingivitis patients showing the lowest levels and each group significantly differing from one another (p < 0.0001 overall). Moreover, this aligns with Buno et al. (1998) [6] and Ghallab et al. (2010) [10], who emphasize IFNγ’s key role in mucosal immunity — particularly in activating macrophages and T cells to defend the oral cavity.

The observed decline in IFNγ among meth users supports findings from Potula et al. (2018) and related studies: methamphetamine suppresses Th1 responses, including IFNγ, impairing adaptive immunity and increasing infection risk [25].

Meanwhile, meth users co-infected with HSV showed even lower IFNγ levels. This agrees with Sainz et al. (2002) [27] and Bigley et al. (2014) [4], who found that HSV can dampen IFNγ-mediated immunity. When combined with meth-induced immunosuppression, the decline becomes more pronounced.

Similarly, gingivitis patients recorded the lowest IFNγ, reflecting a shift away from Th1 responses during chronic inflammation. As noted by Sheibak et al. (2022) [30] and Neurath & Kesting (2024) [23], persistent gingival inflammation often diverts immune signaling toward Th2 or regulatory pathways, reducing IFNγ production over time — even though acute or early periodontitis may temporarily elevate IFNγ.

Thus, methamphetamine use significantly suppresses IFNγ, with HSV co-infection further compounding this suppression. Meanwhile, gingivitis without meth use also results in low IFNγ, though via inflammatory rather than immunosuppressive mechanisms. Ultimately, healthy individuals maintain robust IFNγ output, indicative of intact Th1-mediated immunity and effective mucosal defense.

This study demonstrated that IL-29 levels were highest in patients with gingivitis, followed by methamphetamine users with HSV infection, then meth users without HSV, and lowest in healthy controls. These results are consistent with the findings of Shivaprasad and Pradeep (2015) [32] and Tabari et al. (2021) [35], who reported that elevated IL-29 levels in gingivitis reflect a strong local immune response to bacterial plaque and inflammation. They also showed that IL-29 expression is significantly increased in the gingival tissue of patients with chronic and aggressive periodontitis [32, 35].

Methamphetamine users, especially those co-infected with HSV, also showed higher IL-29 levels than healthy individuals. This may be due to the compounding effect of substance-induced immune dysregulation and viral stimulation. Kevil et al. (2019) [13] and Yang et al. (2020) [42] similarly concluded that chronic methamphetamine use is associated with altered cytokine profiles, including increased interferon production, resulting from tissue injury and prolonged immune activation. Furthermore, Zhou et al. (2011) [44] demonstrated that viral infections like HSV-1 can upregulate IL-29, potentially enhancing antiviral defenses in the oral environment. In fact, IL-29 has been shown to inhibit HSV-1 replication in neuronal cells, supporting its direct antiviral properties [44].

Tognarelli et al. (2019) [39] proposed that IL-29 expression may sometimes be suppressed during viral infections due to immune evasion mechanisms, which could explain fluctuating levels depending on infection stage and host factors [39].

IL-29, a type III interferon, is produced by epithelial cells, dendritic cells, and certain leukocytes in response to microbial and viral stimuli [41]. It binds to a specific receptor complex, activating the JAK-STAT signaling pathway and leading to the expression of interferon-stimulated genes, which enhances antiviral defense and regulates inflammatory responses. In gingivitis, the high IL-29 levels may reflect an attempt to control bacterial invasion and prevent tissue damage. In meth users and meth + HSV cases, chronic inflammation and mucosal injury likely stimulate additional IL-29 production as part of the ongoing immune response [32, 35, 44].

IL-29 also exhibits strong antiviral activity by inducing an antiviral state in infected cells, reducing viral replication, and enhancing immune cell surveillance. It modulates the function of T cells, B cells, dendritic cells, and macrophages, helping to shape immune responses and inflammation [12, 15, 41].

Conclusion

This study concludes that methamphetamine consumption, especially when combined with HSV-1 infection, leads to significant suppression of IFNα and IFNγ, indicating a weakened antiviral and immune response. Conversely, IL-29 levels were markedly elevated in gingivitis and further increased in meth users with HSV-1, which may highlight its potential role as a biomarker for inflammation and viral modulation in the oral cavity.

About the authors

N. A. Alsattar

University of Baghdad

Email: ghada_ibraheem@codental.uobaghdad.edu.iq

MSc in Oral Microbiology/Oral Immunology, Lecturer, Department of Basic Sciences, College of Dentistry

Iraq, Baghdad

Ghada I. Taha

University of Baghdad

Author for correspondence.
Email: ghada_ibraheem@codental.uobaghdad.edu.iq

PhD, Microbiology/Clinical Immunology, Assistant Professor, Department of Basic Sciences, College of Dentistry

Iraq, Baghdad

References

Supplementary files