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IJMSInternational Journal of Molecular Sciences
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4 March 2026

18 Pages

Gene Expression Profiles in the Optic Nerve of Mice with Systemic Acanthamoebiasis

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1
Department of Biology, Parasitology, and Pharmaceutical Botany, Pomeranian Medical University in Szczecin, Powstańców Wielkopolskich 72, 70-111 Szczecin, Poland
2
Institute of Physical Culture Sciences, University of Szczecin, Piastów 40B, 71-065 Szczecin, Poland
3
Department of Physiology, Pomeranian Medical University in Szczecin, Powstańców Wielkopolskich 72, 70-111 Szczecin, Poland
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci.2026, 27(5), 2382;https://doi.org/10.3390/ijms27052382
This article belongs to the Special Issue Molecular Advances in Pathogen Interaction and Host Immunity
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Abstract

Systemic infection with Acanthamoeba spp. can induce inflammatory responses within the visual axis, yet the underlying molecular mechanisms in the optic nerve remain poorly understood. The aim of the study was to determine the gene expression of Nlrp3 (encoding NOD-, LRP- and pyrin domain-containing protein 3, NLRP3), Ptgs2 (encoding cyclooxygenase-2, COX-2), Rela (encoding nuclear factor kappa B, NF-κB), and several cytokines in the optic nerve of mice during disseminated infection with Acanthamoeba sp. (T16 genotype) under various immunological conditions. In immunocompetent mice, Ptgs2 and Ifng expressions were upregulated at the beginning of infection. In the late stages, we found increased levels of Il10 and Nlrp3. In immunosuppressed mice, higher expressions of Nlrp3, Ptgs2, Rela, Il1b, Il10, Il17a, Il21, and Ifng were found in the infected mice compared to the control group. These results indicate that immunosuppression promotes prolonged inflammation by altering innate and adaptive immune responses, contributing to sustained neuroinflammatory processes affecting the optic nerve. This study provides mechanistic insight into host–pathogen interactions in the optic nerve during systemic Acanthamoeba infection. Due to the analysis being based on mRNA expression levels, direct inference regarding protein levels and the actual activity of the investigated immunological pathways is limited.

1. Introduction

The outcome of parasitic infections is determined by the dynamic host–parasite relationship, in which the effectiveness and regulation of the host immune response play a critical role in controlling pathogen survival, tissue invasion, and dissemination [1]. Importantly, the course of infection is strongly influenced by the immunological status of the host, resulting in distinct disease courses in immunocompetent individuals compared with hosts affected by pharmacological immunosuppression or congenital and acquired immunodeficiencies [2]. During infections caused by protozoan parasites, host defense is initiated by innate immune recognition through pattern-recognition receptors, such as Toll-like receptors (TLRs) and cytosolic inflammasomes, which prime early proinflammatory cytokine responses. These pathways integrate with NF-κB-dependent transcriptional programs, resulting in the production of inflammatory mediators [3]. Early innate mechanisms trigger the subsequent activation of adaptive immune pathways mediated by distinct T helper (Th) lymphocyte subsets. Cytokines and chemokines associated with Th1, Th2, and Th17 activities orchestrate pathogen control, immune regulation, and the balance of inflammatory activity [4]. Th1 responses drive cell-mediated immunity, whereas Th17 responses support prolonged inflammatory signaling. Th2-associated cytokines limit tissue damage and maintain immune homeostasis. Therefore, the type of immune response impacts disease progression, infection control, and the risk of dissemination to surrounding tissues [5].
Acanthamoeba is a tiny, free-living protozoan with a cosmopolitan distribution, ubiquitously present across diverse environmental sources [6,7]. Under favorable conditions, they can cause human infection, leading to granulomatous amoebic encephalitis (GAE), Acanthamoeba keratitis (AK), or disseminated infections. Of these manifestations, AK occurs most frequently and is strongly associated with contact lens wear and inadequate lens hygiene [8]. Its pathogenesis is well characterized, with extensive literature detailing host–pathogen interactions at the corneal surface [9]. In contrast, recent experimental data derived from a murine model of disseminated acanthamoebiasis, in which infection was initiated via intranasal aspiration, indicate that Acanthamoeba spp. can also be associated with significant immunological and biochemical alterations in the eye. Ocular tissues exhibited modulation of innate immune responses, reflected by altered expression of Toll-like receptors (TLR2 and TLR4), changes in inflammatory mediator profiles (prostaglandin E2, thromboxane B2) and disruption of antioxidant defense systems, particularly under immunosuppressed conditions [10,11,12]. Although the specific route by which the amoebae reach the eye in the context of systemic infection has not been directly demonstrated in these studies, it has been suggested that Acanthamoeba may reach ocular structures during disseminated infection, potentially involving the optic nerve as a hypothetical migration route from the central nervous system (CNS) [10]. However, this concept remains based on indirect evidence.
The optic nerve is an integral part of the CNS and the visual pathway, making it particularly susceptible to structural and functional damage that can lead to demyelination, axonal injury, and visual impairment [13]. Immune and inflammatory processes have been implicated in the pathogenesis of optic neuritis and other neuroinflammatory disorders, in which activation of innate and adaptive pathways influences cytokine networks and the recruitment of immune cells to neural tissues [14,15]. Specifically, inflammatory signaling cascades, such as NF-κB-dependent transcriptional programs in glial cells, complement activation, and inflammasome complexes, have been shown to drive optic nerve pathology and modulate tissue responses to injury, linking cytokine expression to both cellular damage and repair mechanisms [16,17,18,19]. Therefore, evaluating the expression of genes regulating immune and inflammatory responses in the optic nerve during Acanthamoeba infection provides critical insights into local neuroinflammation processes and host–pathogen interactions. This approach may also reveal molecular pathways that contribute to tissue damage or protection, offering potential therapeutic targets.
This study evaluated the expression of Nlrp3, Ptgs2 (COX-2), Rela (NF-κB), Il17a, Il1b, Il10, Il18, Il21, Il23, Ifng (IFN-γ), Tnf (TNF-α), and Mip2 in the optic nerve of mice infected with Acanthamoeba sp. (T16 genotype) to identify transcriptional profiles of genes associated with pro-inflammatory, anti-inflammatory responses, Th1 and Th17-related cytokines as well as chemokines during disseminated acanthamoebiasis. Investigating the molecular profile of host immune responses offers valuable insights into the process following Acanthamoeba spp. infection.

2. Results

2.1. Expression of Immune and Inflammatory Pathways in the Optic Nerve

The experimental design and the expression of all analyzed immunological and inflammatory genes are shown in Figure 1. Mouse gene symbols are used throughout the manuscript; where relevant, commonly used protein or pathway names are provided in parentheses for clarity.
Figure 1. (I) Experimental design. (II) (a) Fold changes in mRNA expression of Nlrp3, (b) Ptgs2 (COX-2), (c) Rela (NF-κB), (d) Il1b, (e) Il10, (f) Il17a, (g) Il18, (h) Il21, (i) Il23, (j) Ifng, (k) Tnf (TNF-α), and (l) Mip2 in the optic nerve of mice infected with Acanthamoeba sp. (T16 genotype). The mRNA expressions were determined using qRT-PCR. Expression levels were normalized to reference genes Gapdh and β2-microglobulin and are presented as fold change relative to the corresponding control group (2−ΔΔCt). Statistical analyses were performed on ΔCt values. Data represents the means and standard deviations for six experiments. * indicates statistically significant differences, p < 0.05, Mann–Whitney U test. A, immunocompetent Acanthamoeba sp.-infected mice; AS, immunosuppressed Acanthamoeba sp.-infected mice; C, immunocompetent control mice; CS, immunosuppressed control mice; MPS, methylprednisolone sodium succinate.

2.1.1. NLRP3 Inflammasome-Related Genes

The gene expressions of Nlrp3 did not differ significantly in immunocompetent infected (A) and immunosuppressed infected (AS) mice across time points. A significant increase in Nlrp3 mRNA levels was observed in the infected immunocompetent mice compared to uninfected controls at 16 dpi (U = 2, p < 0.01; Figure 1(IIa)). The mRNA level of Il1b remained comparable between infected and uninfected immunocompetent groups throughout the experiment (Figure 1(IId)). In contrast, Il18 expression was significantly lower in the infected group (A) than in controls (C) at the last day of the experiment (U = 3.5, p < 0.05; Figure 1(IIg)).
In immunosuppressed mice, infection was associated with a more pronounced activation of inflammasome-related genes. Significantly higher Nlrp3 expression was observed in the infected animals (AS) compared to uninfected controls (CS) at all analyzed time points: 8 (U = 4, p < 0.05), 16 (U = 5, p < 0.05) and 24 dpi (U = 4, p < 0.05; Figure 1(IIa)). Similarly, Il1b levels were observed in the AS group at 8 dpi, 16 dpi, and 24 dpi (U = 4, p < 0.05; U = 2, p < 0.01; U = 0, p < 0.01, respectively) compared to control mice (Figure 1(IId)). In contrast, no statistically significant differences in Il18 expression were found between infected and uninfected immunosuppressed mice (Figure 1(IIg)).
When comparing immune status, statistically significant differences in mRNA level of Nlrp3 were found between A and AS groups at 8 as well as 16 days post-infection (U = 2, p < 0.01; U = 0, p < 0.01; respectively). A similar pattern was observed for Il1b, with higher expression in the immunosuppressed infected mice at 8 (U = 5, p < 0.05) as well as 16 days post-infection (U = 0, p < 0.01). Additionally, Il18 levels were significantly higher in the AS group at 16 as well as 24 days post-infection (U = 0, p < 0.05; Figure 1(IIg)).

2.1.2. NF-κB and COX-2 Dependent Inflammatory Signaling

In immunocompetent mice, the expression of Rela did not differ between infected and uninfected animals (Figure 1(IIc)). Ptgs2 expression was significantly higher in the A group compared to the C group at the early stage of infection (U = 5, p < 0.05; Figure 1(IIb)).
In immunosuppressed mice, infection resulted in a consistent increase in Rela expression. Higher mRNA levels of Rela were observed in the AS group compared to the CS group at all time points (8 dpi: U = 2, p < 0.01; 16 dpi: U = 5, p < 0.05; 24 dpi: U = 0, p < 0.01; Figure 1(IIc)). Additionally, higher expressions of Ptgs2 were noted in the AS group compared to the CS group at 16 and 24 dpi (U = 4, p < 0.05; U = 5, p < 0.05; respectively; Figure 1(IIb)).
Analysis of immune status revealed statistically significant differences in Ptgs2 mRNA levels between the A and AS groups at all time points, with higher expression observed in immunosuppressed mice (8 dpi: U = 4, p < 0.05; 16 dpi: U = 2, p < 0.01; 24 dpi: U = 0, p < 0.01). No differences in Rela expression were found between infected immunocompetent and immunosuppressed mice.

2.1.3. Th17 Axis-Associated Cytokines

The mRNA level of Il17a in the immunocompetent mice did not differ significantly. There were no differences between the A and C groups at various time points (Figure 1(IIf)). Similarly, no infection-related differences were observed for Il21 and Il23 (Figure 1(IIh,i)).
Gene expression of Il17a in the immunosuppressed mice increased at 16 dpi, and then decreased at 24 dpi (H = 10.61, p < 0.01). Dunn’s post hoc test revealed a difference between 16 dpi and 24 dpi. Comparing infected (AS) and uninfected (CS) mice, higher gene expression was observed in the infected animals at 8 (U = 4, p < 0.05) and 16 dpi (U = 3, p < 0.05; Figure 1(IIf)). Additionally, higher mRNA levels of Il21 were observed in the AS group at 16 dpi (U = 1, p < 0.01) and 24 dpi (U = 0, p < 0.01) compared to the CS group (Figure 1(IIh)). No differences in Il23 expression were observed between infected and uninfected immunosuppressed animals at all analyzed time points (Figure 1(IIi)).
Analysis of immune status revealed statistically significantly higher expression of Th17-related cytokines in the AS group compared to the A group. Significant differences were observed for Il17a at all time points (8 dpi: U = 2, p < 0.01; 16 dpi: U = 0, p < 0.01; 24 dpi: U = 4, p < 0.05), as well as for Il21 and Il23 at 16 dpi (U = 0, p < 0.01) and 24 dpi (U = 1, p < 0.01).

2.1.4. Th1 Axis-Associated Cytokine

The expression of Ifng in the immunocompetent Acanthamoeba sp.-infected mice decreased at 16 dpi, and then increased at 24 dpi (H = 6.636, p < 0.05). Analysis with Dunn’s test showed a difference between 16 dpi and 24 dpi. In the A group, there was higher Ifng expression compared to the C group at the beginning of infection (U = 5, p < 0.05; Figure 1(IIj)). At 16 and 24 dpi, there were no statistically significant differences between the A and C groups. In the immunosuppressed animals, on the other hand, we observed a difference between infected and uninfected mice only on the last day of infection (U = 0, p < 0.01; Figure 1(IIj)).
We also observed a difference between the A and AS groups at 16 dpi (U = 0, p < 0.01). Higher gene expression was observed in the AS group.

2.1.5. Regulatory and Effector Mediators

The gene expressions of Il10 were statistically different between the infected and uninfected immunocompetent groups at 16 (U = 0, p < 0.01) and 24 dpi (U = 1, p < 0.01); expressions were higher in the A group compared to the C group (Figure 1(IIe)). No changes in Tnf (encoding TNF-α) expression were observed in immunocompetent infected mice at any time point (Figure 1(IIk)). Similarly, Mip2 expression remained stable during the course of infection (Figure 1(IIl)).
In immunosuppressed mice, infection resulted in a marked increase in Il10 expression at all analyzed time points, with significantly higher mRNA levels detected in the infected group (AS) compared to the control one (CS) (8 dpi: U = 5, p < 0.05; 16 dpi: U = 0, p < 0.01; 24 dpi: U = 1, p < 0.01; Figure 1(IIe)). The gene expressions of Tnf and Mip2 in the AS and CS groups did not differ (Figure 1(IIk,l)). When comparing immune status, a statistically significant difference in Il10 mRNA levels was found between the A and AS groups at day 16 post-infection (U = 0, p < 0.01).
Despite the use of four different primer pairs, no expression of Il6 was detected in the optic nerve of either control or infected animals. This was observed despite good RNA quality, stable amplification of reference genes, and consistent results across technical duplicates. Some primers were designed in-house, whereas others were adopted from published studies [20]. As no external spike-in control was included to assess reaction efficiency, the presence of PCR inhibitors in the RNA samples or reduced amplification efficiency specific to Il6 cannot be completely ruled out.

2.2. Clustering Analysis

Global heatmap analysis provided an overview of immune-related gene expression patterns in the optic nerve across experimental groups and time points following Acanthamoeba infection (Figure 2a). Gene expression data were normalized to control groups and visualized using row-wise scaling, enabling comparison of relative expression dynamics within individual genes across experimental conditions. Rows represent individual cytokines, while columns correspond to experimental groups. Color intensity reflects relative cytokine levels (row-scaled z-scores), with red indicating lower-than-average values, black indicating average values, and green indicating higher-than-average cytokine expression. The heatmap revealed heterogeneous expression profiles among the analyzed cytokines, reflecting time-dependent and group-specific modulation of the immune response. A dendrogram shown on the left side of the heatmap illustrates the results of agglomerative hierarchical clustering based on the similarity of cytokine profiles across experimental conditions. Cytokines that cluster closely together exhibit similar response patterns, suggesting potential co-regulation or involvement in related immunological pathways.
Figure 2. Heatmap analysis of immune-related gene expression in the optic nerve. (a) Global heatmap showing the relative expression of immune-related genes across experimental groups. (b) Functional heatmap depicting composite immune response indices calculated from cytokine gene expression data, including the Th17/Th1 balance, pro-/anti-inflammatory index, and inflammasome/anti-inflammatory index. A, immunocompetent Acanthamoeba sp.-infected mice; AS, immunosuppressed Acanthamoeba sp.-infected mice; C, immunocompetent control mice; CS, immunosuppressed control mice; Numeric suffixes (8, 16, 24) indicate days post-infection. For example, A8, A16, and A24 represent immunocompetent Acanthamoeba sp.-infected mice analyzed at 8, 16, and 24 days post-infection, respectively.
Functional heatmap analysis (Figure 2b), based on calculated immune balance indices, including Th17/Th1 balance, pro-/anti-inflammatory index and inflammasome/anti-inflammatory index, demonstrated clear time-dependent shifts in immune regulation. These indices indicated a progressive bias toward pro-inflammatory and inflammasome-driven responses in infected groups, particularly at later time points. Unlike the global gene expression heatmap, the functional heatmap was generated without additional scaling to preserve the biological interpretability of the calculated indices. Collectively, these heatmap analyses illustrate dynamic and condition-dependent immune response patterns during Acanthamoeba infection and provide a global context for subsequent quantitative and gene-specific analyses.

2.3. Gene Ontology and Pathway Analysis

To further characterize biological processes and signaling pathways associated with changes in immune- and inflammation-related gene expression in the optic nerve following Acanthamoeba infection, Gene Ontology (GO) biological process and Reactome pathway enrichment analyses were performed. For visualization, the top 10 significantly enriched terms from each analysis were selected independently for immunocompetent and immunosuppressed mice and subsequently combined into mirrored bar charts to enable comparison between groups.
In immunocompetent mice, statistically significant differences in gene expression were detected for Nlrp3, Ptgs2, Ifng, and Il10. GO biological process analysis revealed significant associations with terms related to regulation of inflammatory processes, positive regulation of cytokine production, and positive regulation of immune response (Figure 3a). Reactome pathway analysis identified gene and protein expression mediated by JAK–STAT after IL-12 stimulation, as well as IL-10 and IL-12 signaling (Figure 3b). In immunosuppressed mice, statistically significant changes in gene expression were observed for Nlrp3, Il1b, Ptgs2, Rela, Il17a, Il21, Ifng, and Il10. GO biological process analysis identified terms associated with regulation of inflammatory response, positive regulation of cytokine production, and positive regulation of gene expression (Figure 3a). Reactome pathway analysis demonstrated signaling related to signaling by interleukins and cytokine signaling in the immune system (Figure 3b). Distinct profiles of biological processes and signaling pathways were observed between immunocompetent and immunosuppressed mice following Acanthamoeba infection.
Figure 3. GO biological processes (a) and Reactome pathways enrichment (b) in the optic nerve of immunocompetent and immunosuppressed Acanthamoeba sp.-infected mice. Differentially expressed immune- and inflammation-related genes were analyzed in immunocompetent and immunosuppressed mice. GO enrichment analysis identified the top biological processes, and Reactome pathway analysis highlighted associated signaling pathways. Bar height represents –log10 (p-value) for each term/pathway. Group A, immunocompetent Acanthamoeba sp.-infected mice, Group AS, immunosuppressed Acanthamoeba sp.-infected mice.

3. Discussion

Inflammation is a fundamental host response to a wide range of damaging stimuli, including infections, tissue injury, and various forms of cellular stress. Early inflammatory signals trigger molecular cascades that activate immune mechanisms, enabling the elimination of harmful factors but also contributing to the pathogenesis of chronic neurodegenerative processes when dysregulated. In recent years, increasing attention in basic and translational research has been devoted to the role of the NLRP3 inflammasome as a central regulator of inflammatory initiation. The NLRP3 inflammasome is a multiprotein complex that, upon activation, promotes caspase-1–dependent maturation of the pro-inflammatory cytokines IL-1β and IL-18, thereby modulating both innate and adaptive immune responses [21,22].
Experimental models have demonstrated that activation of the NLRP3 inflammasome is associated with degenerative processes in the optic nerve and retina, whereas inhibition of this pathway attenuates the inflammatory cascade and preserves visual signal conduction [23]. Experimental models of optic nerve injury, such as optic nerve crush, have shown that NLRP3 activation in microglial cells is linked to retinal ganglion cell (RGC) degeneration and compromised axonal integrity [24]. Furthermore, in models of glaucoma and neuroinflammatory conditions affecting the optic nerve, suppression of NLRP3 activity reduced neurodegeneration and RGC death, highlighting NLRP3 as a potential therapeutic target in optic neuropathies [25].
In our study of immunocompetent mice infected with Acanthamoeba sp., an increase in Nlrp3 expression was observed at day 16 post-inoculation; however, no concomitant activation of the Il1b or Il18 genes was detected. At day 24 post-infection, a decrease in Il18 expression was noted in this group. This expression pattern is consistent with NLRP3 priming without full inflammasome activation, indicating a regulated inflammatory response in immunocompetent hosts. In contrast, immunosuppressed mice exhibited increased expression of both Nlrp3 and Il1b at all examined time points. Histological analysis of the retina in these animals revealed increased thickness of the outer nuclear layer and enhanced vacuolization within the outer plexiform layer, changes that were absent in the other experimental groups [11]. These findings suggest that full activation of the NLRP3 inflammasome under immunosuppressive conditions may contribute to exacerbated inflammation and structural alterations associated with optic nerve involvement during disseminated infection.
A central mechanism regulating NLRP3 priming is the NF-κB signaling pathway, whose key transcription factor is Rela [25,26,27]. In our study, immunocompetent mice did not exhibit significant Rela expression, which may explain the lack of Il1b and Il18 induction despite the observed increase in Nlrp3 expression. In contrast, mice infected with Acanthamoeba sp. and subjected to immunosuppression displayed Rela expression at all examined time points, suggesting dysregulation of the NF-κB pathway and sustained activation of pro-inflammatory gene transcription. Together, these findings suggest that NF-κB–dependent transcription may facilitate the transition from NLRP3 priming to full inflammasome activation under immunosuppressive conditions. Although direct comparisons between immunosuppressed and immunocompetent infected mice did not reveal statistically significant differences in Rela expression, a decrease was noted in the immunosuppressed control group. This reduction is most likely attributable to the effects of methylprednisolone (the drug used to attenuate the immune response in the AS and CS groups), which is known to inhibit the activity of NF-κB family proteins [28].
Following activation of early inflammatory mechanisms, the subsequent course of the immune response depends on engagement of the adaptive immune system. A particularly important role is played by Th17 lymphocytes and their associated cytokines (IL-17A, IL-21, and IL-23) [29,30]. The Th17 axis is critical for maintaining the balance between effective pathogen clearance and protection of tissues from excessive damage, including structures of the visual axis such as the optic nerve and retina [31]. In our study, expression of genes associated with the Th17 axis displayed a differential pattern depending on the immune status of the mice. In immunocompetent animals, expression levels of Il17a, Il21, and Il23 did not show a significant increase at any time point, suggesting a lack of Th17 axis activation during the course of infection. In contrast, immunosuppressed mice exhibited increased Il17a and Il21 expression without Il23 upregulation. The lack of correlation with IL-23 argues against full engagement of the classical Th17 activation pathway [32], potentially contributing to prolonged and chronic inflammation [33,34].
A smooth transition from the Th17 response to the Th1 axis reflects the subsequent direction of immunoregulation during the course of infection. The Th1 axis, characterized primarily by the synergistic action of IL-18 and IL-12 leading to IFN-γ production, is responsible for cell-mediated immunity directed against intracellular parasitic infections [35]. Analysis of Th1-axis markers revealed that the development of the cellular immune response is strongly dependent on the host’s immunological status. In immunocompetent mice infected with Acanthamoeba sp. (genotype T16), an early increase in Ifng gene expression was observed, indicating rapid activation of a Th1-type response and engagement of cellular effector mechanisms. In contrast, in immunosuppressed hosts infected with the amoeba, increased Ifng expression was detected only at the final stage of observation, accompanied by reduced Il18 expression. This profile suggests delayed or partially dysregulated Th1 activation alongside chronic Th17 activity, indicating a shift toward a prolonged inflammatory state that may exacerbate optic nerve damage. Despite the lack of IL-12 assessment, this pattern is consistent with models of chronic infection and neuroinflammatory diseases, in which sustained inflammation promotes neural tissue degeneration [29,30].
During inflammation affecting structures of the visual axis, precise regulation of the immune response is crucial, as excessive or prolonged activation of inflammatory mechanisms may lead to irreversible damage to the optic nerve [36]. In this context, interleukin-10 plays an important neuroprotective role by limiting the intensity of the inflammatory response and protecting neural tissue from secondary immune-mediated injury [37,38]. Available data indicate that IL-10 plays a significant immunoregulatory role during parasitic infections. In cerebral toxoplasmosis, increased IL-10 expression has been shown to be critical for limiting excessive Th1-type inflammatory responses by suppressing pro-inflammatory cytokine production and reducing inflammatory cell infiltration into the brain. Absence of IL-10 leads to exacerbated neuroinflammation and more severe histopathological changes in the CNS [39]. Similar mechanisms have been observed in other neurotropic parasitic infections, in which IL-10 restricts excessive inflammation and effector cell influx into the CNS. At the same time, the immunosuppressive nature of this cytokine may facilitate parasite persistence within the CNS and promote the development of chronic infection [40,41,42].
In studies examining Il10 gene expression in animals experimentally infected with Acanthamoeba sp. (genotype T16), increased expression was detected in both immunocompetent and immunosuppressed hosts, indicating that activation of regulatory mechanisms represents a common host response to Acanthamoeba infection regardless of immune status. In our model, elevated Il10 expression may have been modulated by Toll-like receptor activation [43], consistent with previously observed changes in Tlr2 and Tlr4 expression in the eyeballs of mice from the same model [10]. Furthermore, in immunocompetent mice, early induction of Ifng expression was observed, followed by a later increase in Il10, while Tnf remained undetectable. This profile suggests an initially moderate Th1-type pro-inflammatory response that does not result in intense optic nerve inflammation associated with disseminated Acanthamoeba infection. At later stages of infection, regulatory mechanisms become activated, limiting excessive Th1/Th17 activity and protecting optic nerve tissue from immune-mediated damage. In contrast, in immunosuppressed animals, Il10 expression increased earlier and persisted throughout the observation period, whereas Ifng upregulation was delayed and occurred only at later stages of infection, and Tnf remained undetectable. This pattern suggests that IL-10 may simultaneously inhibit effective parasite clearance, thereby promoting a chronic, low-grade inflammatory state.
In the effector phase of the inflammatory response within the optic nerve, mediators responsible for maintaining and modulating the local inflammatory microenvironment play a crucial role. These include pro-inflammatory cytokines such as TNF-α, chemokines that recruit inflammatory cells (including MIP-2/CXCL2), and inflammation-induced enzymes, particularly cyclooxygenase-2 (COX-2), encoded by the Ptgs2 gene [44]. Numerous models of optic neuritis and other neuroinflammatory diseases have demonstrated that excessive activation of these mediators contributes to blood–nerve barrier disruption, microglial activation, increased oxidative stress, and progression of demyelination and axonal degeneration [45,46,47]. Particular importance in chronic neuroinflammatory processes is attributed to the COX-2 pathway, whose activation leads to increased synthesis of bioactive eicosanoids such as prostaglandin E2 (PGE2) and thromboxane B2 (TXB2). These mediators exert strong immunomodulatory and vascular effects, influencing vascular permeability, blood flow regulation, glial cell activation, and the maintenance of chronic inflammation within CNS structures, including the visual axis [48,49]. The relevance of this pathway in Acanthamoeba spp. infections is supported by studies demonstrating significant changes in PGE2 and TXB2 concentrations in ocular structures of both immunocompetent and immunosuppressed mice, indicating activation of the COX-2 axis as an important component of the local inflammatory response [12]. In our study, the expression profile of Ptgs2, encoding COX-2, differed depending on the host’s immunological status. In immunocompetent mice, increased expression was observed during the early phase post-infection, likely reflecting transient activation of inflammatory mechanisms that initiate host defense and tissue adaptation. In contrast, in immunosuppressed mice, Ptgs2 expression was enhanced at later stages of infection, indicating persistence of chronic inflammation. Prolonged activation of the COX-2 axis and the associated production of PGE2 and TXB2 may represent a mechanism contributing to the gradual optic nerve involvement during systemic Acanthamoeba infection, rather than direct parasite invasion. At the same time, no significant increase in Tnf or Mip2 gene expression was detected in either immunocompetent or immunosuppressed mice, suggesting the absence of a robust, acute TNF-dependent inflammatory response and limited neutrophil recruitment to optic nerve tissue, likely due to a lack of MIP-2 induction. This observation may be linked to elevated IL-10 levels detected at nearly all time points across experimental groups, as IL-10 is known to suppress TNF-α expression and neutrophil-attracting chemokines, including MIP-2 [50,51,52]. In this context, the lack of substantial TNF-α pathway activation is consistent with reports indicating that certain parasitic infections may proceed without full engagement of this mediator, while maintaining a regulated, TNF-independent inflammatory response modulated by anti-inflammatory cytokines and lipid mediators [53].
The observed transcriptional changes may reflect either direct local parasite-associated responses or secondary neuroinflammatory processes resulting from systemic infection. Several limitations of this study should be acknowledged. Due to the limited sample size, our analyses were restricted to mRNA expression, and the protein-level validation was not performed, which limits the ability to directly infer functional activity of the investigated pathways, particularly NLRP3 inflammasome activation. Histopathological evaluation of the optic nerve was not possible in this study due to tissue unavailability, preventing direct evaluation of immune cell infiltration and parasite localization. Although retinal histopathology has been previously reported [11], future studies incorporating immunohistochemistry or other tissue-based approaches would be valuable to directly correlate cellular infiltration with transcriptional changes observed in the optic nerve. In addition, IL-12 expression was not assessed, which limits the interpretation of Th1 pathway activation and its interaction with the Th17 axis. The undetectable IL-6 expression suggests that certain inflammatory signals may be limited or absent in the optic nerve during infection, potentially reflecting tissue-specific regulation of cytokine expression. The lack of spike-in inhibition controls to monitor amplification efficiency represents a technical limitation, as the potential presence of PCR inhibitors or gene-specific amplification issues cannot be entirely excluded. Finally, although the murine model provides valuable mechanistic insights, extrapolation to human Acanthamoeba infection should be done with caution due to potential interspecies differences in immune regulation. Future studies addressing these limitations, including protein-level analyses and direct parasite localization, would strengthen the understanding of optic nerve immunopathology during systemic acanthamoebiasis.

4. Materials and Methods

4.1. Parasite

The experimental infection was carried out using Acanthamoeba sp. strain AM22 (genotype T16; GenBank accession no. GQ342607.1), originally isolated from a bronchoaspirate of a hemato-oncology patient [54]. Amoebae were cultivated in vitro by serial passaging on non-nutrient agar plates supplemented with heat-inactivated Escherichia coli at 37 °C.
This clinical strain was chosen for this study because it is associated with pulmonary and systemic infections, rather than keratitis. Its characteristics make it suitable for modeling systemic infection and investigating pathways of parasite dissemination.

4.2. Murine Model

The murine model applied in the present study has been described in detail in our previous publications [10,55]. Briefly, experiments were performed using male BALB/c mice aged 6–10 weeks (n = 96; mean body weight 23 g), obtained from the Centre of Experimental Medicine at the Medical University of Białystok (Poland). Animals were maintained under standard laboratory conditions with controlled temperature and unrestricted access to food and water. Mice were randomly allocated into four experimental groups based on infection and immune status: immunocompetent infected (A, n = 30), immunosuppressed infected (AS, n = 30), immunocompetent non-infected controls (C, n = 18), and immunosuppressed non-infected controls (CS, n = 18).

4.2.1. Immunosuppression Model

Immunosuppression in the AS and CS groups was induced by intraperitoneal administration of methylprednisolone sodium succinate (Solu-Medrol; Pfizer, New York, NY, USA) at a volume of 220 µL per animal. This glucocorticoid acts at both genomic and non-genomic levels, transiently modulating immune function. At the genomic level, it binds intracellular glucocorticoid receptors and translocates to the nucleus, where it inhibits transcription of pro-inflammatory genes and promotes anti-inflammatory gene expression. At the cellular level, methylprednisolone reduces the activity and number of T and B lymphocytes, monocytes/macrophages, neutrophils, and eosinophils, resulting in reversible suppression of systemic immune responses. Non-genomic effects occur rapidly and include modulation of membrane receptors and ion channels [56]. This treatment was used as a pharmacological model of immunosuppression, primarily targeting inflammatory and cytokine-mediated immune responses rather than inducing a permanent immune-deficient state. Immunological parameters, including changes in peripheral blood cell populations, have been previously reported [57], confirming the systemic effect of the treatment.

4.2.2. Experimental Infection

Infection was induced via intranasal inoculation of 3 µL of a saline suspension containing 10,000–20,000 Acanthamoeba trophozoites. Control animals received intranasal administration of pure saline (3 µL). Animals were euthanized at three time points post-infection corresponding to the early (day 8), intermediate (day 16), and late (day 24) stages of infection. At each time point, ten animals from the infected groups and six animals from each control group were sacrificed by intraperitoneal injection of sodium pentobarbital (Euthasol vet; 2 mL/kg body weight). Immediately after euthanasia, optic nerves were collected, rapidly frozen, and stored at −80 °C until further analyses.
Infection was confirmed by re-isolation of amoebae from multiple organs, including the entire eyeball and optic nerve, by culturing on non-nutrient agar supplemented with bacteria and incubated at 41 °C for 10 days. The results of amoebal re-isolation from all examined organs are summarized in Supplementary Table S1, and representative culture images are presented in Supplementary Figure S1. However, no trophozoites were detected in histological sections of the eyeballs, indicating that the local parasite burden may have been low or unevenly distributed.

4.3. Gene Expression

Frozen mouse optic nerve samples were mechanically homogenized, after which total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol. The yield and quality of the isolated RNA were assessed spectrophotometrically with a NanoDrop ND-1000 instrument (Thermo Fisher Scientific, Waltham, MA, USA). Only RNA samples of acceptable purity were used for downstream analyses.
Gene expression analysis was carried out using a two-step quantitative real-time PCR approach. Relative mRNA levels of the target genes were calculated by normalization to the mean expression of two reference genes, Gapdh and β2-microglobulin. Stable amplification of both reference genes was confirmed across all samples. For cDNA synthesis, 1 µg of total RNA was reverse-transcribed using the First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA; cat. no. K1612) with oligo(dT) primers (Table 1). Quantitative PCR reactions were performed on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using Power SYBR Green PCR Master Mix (Applied Biosystems; cat. no. A25918). Each sample was analyzed in duplicate, and mean values were used for subsequent statistical evaluation. No-template controls (NTC) were included in each run and showed no amplification. Spike-in/inhibition controls were not performed. Real-time PCR was carried out using the following cycling conditions: 95 °C for 15 s, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Melting curve analysis confirmed the amplification of a single PCR product.
Table 1. Primers used in qRT-PCR (B2M, Beta-2 microglobulin).
Relative gene expression was calculated using the comparative Ct (2−ΔΔCt) method. The comparative Ct approach assumes comparable amplification efficiencies between target and reference genes. Reaction efficiencies were not determined using standard curves.

4.4. Gene Ontology and Pathway Analysis

Pathway analysis and functions of genes in molecular, cellular and biological processes were performed using the site address https://maayanlab.cloud/Enrichr/ (accessed on 7 February 2026).

4.5. Statistical Analysis

Initially, statistical analyses were performed using StatSoft Statistica 8.0 and GraphPad 4.0. For each of the studied parameters, the arithmetic mean and the standard deviation from the arithmetic mean (SD) were calculated. Due to the fact that the data did not follow normal distributions (according to the Shapiro–Wilk test), differences between the studied parameters were calculated by using the non-parametric Mann–Whitney U-tests (U) and Kruskal–Wallis tests (H) followed by Dunn’s post hoc tests. A p-value of less than 0.05 was considered statistically significant. Heatmaps were generated using the Heatmapper web-based tool (heatmapper.ca; accessed on 15 January 2026).

5. Conclusions

Systemic Acanthamoeba infection triggers complex immune responses involving the optic nerve, which are strongly shaped by the host’s immune status. In immunocompetent mice, NLRP3 inflammasome priming occurs without full activation, accompanied by moderate Th1 responses and controlled IL-10 expression, consistent with limited inflammation and relative preservation of retinal and optic nerve structure. In contrast, immunosuppressed mice exhibit sustained NF-κB signaling, transcriptional profile consistent with NLRP3 inflammasome activation, elevated Th17 cytokines, and delayed Th1 responses, creating a prolonged pro-inflammatory environment that correlates with structural alterations in the retina. Elevated Il10 and Ptgs2 expressions in these mice suggest simultaneous engagement of regulatory and eicosanoid-mediated pathways, which modulate but do not prevent chronic neuroinflammation. Together, these findings provide new insights into immune mechanisms associated with optic nerve involvement during systemic Acanthamoeba infection under different host immune conditions.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27052382/s1.

Author Contributions

Conceptualization, K.K.; methodology, N.Ł.-A., D.K.-B. and K.K.; validation, K.K.; formal analysis, I.M.W. and K.K.; investigation, I.M.W., P.T. and M.C.; resources, P.T. and M.C.; data curation, K.K.; writing—original draft preparation, I.M.W. and K.K.; writing—review and editing, I.M.W. and K.K.; visualization, I.M.W.; supervision, K.K.; project administration, K.K.; funding acquisition, K.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was approved by Ethics Committee for Scientific Experiments on Animals in Szczecin (No. 29/2015, dated 22 June 2015) and Poznań (No. 64/2016, dated 9 September 2016).

Data Availability Statement

Derived data supporting the findings of this study are available from the corresponding author (K.K.) on request.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AImmunocompetent Acanthamoeba sp.-infected mice
AKAcanthamoeba keratitis
ASImmunosuppressed Acanthamoeba sp.-infected mice
B2MBeta-2-microglobulin
CImmunocompetent uninfected mice
CNSCentral nervous system
CSImmunosuppressed uninfected mice
dpidays post-infection
GOGene ontology
IL-1βInterleukin 1β
IL-10Interleukin 10
IL-17AInterleukin 17A
IL-18Interleukin 18
IL-21Interleukin 21
IL-23Interleukin 23
INF-γInterferon γ
MIP-2Macrophage Inflammatory Protein 2
RGCRetinal ganglion cell
SDStandard deviation
Th1T helper 1 cells
Th17T helper 17 cells
TNF-αTumor necrosis factor α

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