Cytokine gene expression in feline leishmaniasis: why cats might be less clinically affected than dogs

TL;DR

This study shows that cats infected with Leishmania infantum mount a strong Th1 immune response, which may explain why they show milder symptoms compared to dogs.

Contribution

The study provides the first evidence of a Th1-like cytokine response in feline macrophages infected with L. infantum.

Findings

Feline macrophages showed upregulated IL-2, IFN-γ, and TNF-α at 72 hours post-infection.

IL-4, IL-6, and IL-10 remained stable or slightly downregulated, indicating a Th1-type response.

The Th1-like response may help cats control L. infantum infection more effectively than dogs.

Abstract

Canine leishmaniasis caused by Leishmania infantum is recognized as one of the most important neglected vector-borne diseases of zoonotic concern worldwide. Cats may also be infected with L. infantum, though the mechanisms underlying the immune response to this protozoal infection in the feline host remain poorly understood. In this study, the early cytokine gene-expression profile was investigated by in vitro infection of feline monocyte-derived macrophages with L. infantum.

Primary macrophages were matured from peripheral blood mononuclear cells of a healthy domestic cat, and cells were collected at different time points post-infection (i.e., 4, 24, and 72 h) for microscopic evaluation and quantitative real-time PCR analysis of cytokine expression [interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α].

Infection rates ranged from 35.6% to 48.2%, with 2.1–2.4 intracellular parasites per infected cell. Gene-expression analysis revealed a marked upregulation of IL-2, IFN-γ, and TNF-α at 72 h, indicating a predominant Th1-type pro-inflammatory response, while IL-4, IL-6, and IL-10 remained stable or slightly downregulated.

These findings provide unprecedented evidence that feline macrophages activate a Th1-like cytokine pattern following in vitro infection with L. infantum. Overall, the outcomes of this investigation support the hypothesis that, when compared with dogs, the milder clinical course of leishmaniasis in cats is related to a predominant Th1-like immune response, eventually contributing to the infection control in naturally infected animals.

The online version contains supplementary material available at 10.1186/s13071-026-07285-5.

Figures

Feline monocyte-derived macrophages infected with *Leishmania infantum* at 4 h (A) and 72 h (B)

Relative mRNA gene expression (relative units) of cytokines IL-2, IFN-γ, TNF-α, IL-4, IL-6, and IL-10 from feline monocyte-derived macrophages infected by *L. infantum* at 4, 24, and 72 h in triplicate (standard deviations shown). Asterisks indicate the statistical difference between timepoints (P* < 0.05, *P* < 0.01, P* < 0.001). Adjusted *P*-values are detailed in the textFig. 4Cytokine expression in feline leishmaniasis. After macrophages phagocytize *L. infantum* promastigotes (in vivo after sand fly blood meal or in vitro after experimental infection of monocyte-derived macrophages), and

Full text

Background

Leishmaniases are vector-borne neglected tropical diseases caused by protozoa of the genus Leishmania (Kinetoplastida, Trypanosomatidae), with over 350 million people at risk of infection, and more than 1 million reported cases worldwide [1–3]. More than 20 Leishmania species may infect a wide range of vertebrate hosts, including humans, by replicating within macrophages and dendritic cells and causing immunological disorders with the production of circulating immune complexes (CICs), which lead to clinical manifestations ranging from self-healing cutaneous lesions to fatal visceral disease [4]. Although leishmaniasis is endemic mainly in low-income regions of East Africa, Southeast Asia, and Latin America, the disease also represents a persistent public health issue in the Mediterranean basin [5].

Leishmania infantum is the main causative agent of visceral leishmaniasis in the Mediterranean and parts of South America. This zoonotic species is transmitted by phlebotomine sand flies [6], with domestic dogs as the primary reservoirs. However, in regions where infection is endemic, many other animal species may be infected [7]. For example, cats have been reported as a potential host of L. infantum, with cases of cutaneous or oral lesions described with increasing frequency across the Mediterranean basin and Brazil [8–13]. In addition, xenodiagnosis studies have demonstrated that naturally infected cats can transmit L. infantum to sand flies under experimental conditions. In Italy, Maroli et al. [14] showed that L. infantum could develop in Phlebotomus perniciosus after feeding on a naturally infected cat. Similarly, cats naturally infected with L. infantum in Brazil were demonstrated to infect Lutzomyia longipalpis sand flies that fed on them [15].

The seroprevalence of leishmaniasis in domestic cats in the Mediterranean basin ranges from 12.6% in Italy to 24.7% in Portugal, with intermediate rates in Greece (23.2%), Israel (16.6%), Spain (15%), and France (13.3%) [13]. Nonetheless, cats are considered secondary hosts, rather than reservoirs, and often remain subclinically infected or develop only mild, nonspecific clinical signs (e.g., mild weight loss, reduced appetite, lethargy, lymph node enlargement, occasional oral or ocular lesions, and alopecia) [12].

Despite the differences in clinical presentation in feline hosts compared with those in infected dogs, the immune response against L. infantum in cats remains scarcely understood. Much more is known in dogs, where a dominant Th1-type response characterized by the production of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12) is associated with protection against L. infantum infection [16–18]. These cytokines activate macrophages toward an M1 phenotype, promoting intracellular parasite killing. Conversely, disease progression is correlated with a shift toward Th2/regulatory cytokines, including IL-4, IL-6, and IL-10, which prompt antibody production, suppress macrophage activation, and favor parasite survival [19, 20]. In vitro studies on canine monocyte-derived macrophages demonstrated that infection by L. infantum induces strong expression of IL-4 and IL-6, whereas co-infection with the nonpathogenic L. tarentolae decreases such anti-inflammatory cytokines, increasing IL-12 transcription [18].

Dogs generally display higher levels of anti-Leishmania spp. antibodies and greater IFN-γ production than cats, suggesting that humoral and cell-mediated immunity against L. infantum is overall lower in cats than in dogs [12, 21, 22]. However, a deep understanding of feline cytokine responses during the early stages of L. infantum infection is still lacking, and scientific data on the immune responses to Leishmania spp. using peripheral blood mononuclear cells (PBMCs) or macrophages are limited in cats [21, 23]. Hence, most current knowledge about feline immune responses to Leishmania spp. derives from observations in naturally infected animals, in which mixed Th1/Th2 cytokine profiles and variable IFN-γ and IL-10 expression levels have been reported [24, 25]. However, these studies might be biased by the clinical conditions of patients, which may affect cytokine production. Therefore, in vitro assessments that can define immunological mechanisms occurring in cats are needed. In the present study, the early cytokine gene-expression profile of feline monocyte-derived macrophages was characterized following experimental infection with L. infantum.

Methods

Parasites and culture conditions

Leishmania infantum promastigotes at the third passage (MCAN/IT/2025/Max; University of Bari, Italy), isolated from a naturally infected dog, were cultivated in Schneider’s insect medium (Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10% human urine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 26 °C, pH 7.4.

Animal and blood collection

A clinically healthy 3-year-old European shorthair domestic cat was enrolled as a blood donor in this study with written informed consent from the owner. The cat was clinically healthy, with unremarkable physical examinations and normal hematological, biochemical, and urinary evaluations. The animal tested negative for feline immunodeficiency virus (FIV) antibody, feline leukaemia virus (FeLV) antigen, and Leishmania infection by both immunofluorescence antibody test (IFAT) serology, and PCR, as well as for other parasitic pathogens. Peripheral blood samples (6 mL) were collected via jugular venipuncture into Vacutainer™ K3-EDTA-coated tubes. In order to avoid further interference caused by biological factors (e.g., age, nutrition, gender), all samples were obtained from the same donor. Indeed, by focusing on relative gene expression in isolated cells, this approach minimizes the influence of individual biological variability. The study protocol was approved by the ethical committee of the Department of Veterinary Medicine of the University of Bari, Italy (Prot. Uniba 56/2024). The methods were carried out in accordance with international, national, and/or institutional guidelines and regulations for handling animal samples.

Isolation of primary feline monocyte-derived macrophages

Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation with Lympholyte® (Cedarlane, Canada), following the protocol described in Louzada-Flores et al. [26] with some modifications [23]. Briefly, the blood was mixed 1:1 with warm RPMI-1640 medium (Gibco, USA) and carefully layered over twice the volume of Lympholyte® (Cedarlane, Canada) in 15 mL conical tubes. Samples were centrifuged at 800 × g for 45 min at room temperature. The buffy coat layer was collected and transferred to new tubes, where cells were washed twice in warm RPMI-1640 at 400 × g for 10 min. Viable cells were counted in a Neubauer chamber by trypan blue exclusion, and resuspended in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 100 ng/mL of recombinant mouse colony-stimulating factor 1 (CSF-1; ImmunoTools) to favor the maturation of macrophages. Cells were seeded in 6-well plates (for RNA extraction) or 24-well plates containing sterile glass coverslips (for microscopy) at a concentration of 1 × 10^6^ cells/mL and 2 × 10^5^ cells/mL, respectively, and incubated at 37 °C with 5% CO_2_ for 3 days. On the 4th day, the medium was replaced to remove nonadherent cells, and the plate was kept until the 6th day for maturation of macrophages with CSF-1.

Infection of feline monocyte-derived macrophages by Leishmania infantum

Prior to infection, promastigotes at the late stationary phase (5th day of culture) were washed three times in sterile phosphate-buffered saline (PBS), resuspended in RPMI-1640 medium, and the concentration was adjusted to a multiplicity of infection (MOI) of 10:1 (parasites:cell). A volume of 500 μL for 6-well plates and 200 μL for 24-well plates of parasites was added to the wells according to the MOI and then incubated for 4 h at 37 °C and 5% CO_2_ to allow parasite internalization. Subsequently, the wells were washed three times with PBS to remove noninternalized parasites. Cells were scraped and collected at 4, 24, and 72 h post-infection, and the pellets were stored at −80 °C until RNA extraction. Three independent biological replicates with two technical replicates (wells) were analyzed per condition. Uninfected cells served as negative controls. For each condition and timepoint, the percentage of infected cells and the mean number of intracellular parasites per cell were determined by microscopic examination of Diff-Quik-stained coverslips by counting 200 cells.

RNA extraction and cDNA synthesis

Total RNA was extracted from scraped monocyte-derived macrophages using the RNeasy^®^ Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol, and the concentration and purity were determined using a spectrophotometer (NanoDrop, Thermo Scientific). First-strand cDNA was synthesized from 180 ng total RNA using the SuperScript IV VILO™ Master Mix (Thermo Fisher Scientific, USA) following the manufacturer’s instructions.

Quantitative real-time PCR

Quantitative real-time PCR (qPCR) was performed to assess the expression of feline cytokines IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α, using β-tubulin as the housekeeping gene. Primers (400 nM) and probes (250 nM) were selected from previous studies [27] and are reported in Table 1. Amplification reactions were conducted in a CFX96 thermocycler using SSO Advanced mix (Bio-Rad, USA). Relative gene expression was calculated using the 2^−ΔΔCt^ method [28], normalized to the housekeeping gene, and expressed relative to uninfected controls.

Statistical analysis

Statistical analyses were performed using GraphPad Prism version 10.0 (GraphPad Software, San Diego, CA, USA). Data normality was assessed using the Shapiro–Wilk test. Differences among groups were evaluated by two-way ANOVA followed by Tukey’s post hoc test. Results were considered statistically significant when P < 0.05.

Results

Microscopic observations of quantitative infection parameters (parasite load and infection rate) and Diff-Quik-stained cultures are shown in Figs. 1 and 2, respectively. At 4 h post-infection, L. infantum-infected feline monocyte-derived macrophages showed a mean infection rate of 48.2 ± 4.2%, with 2.1 ± 0.4 parasites per infected cell (Figs. 1 and 2A). At 24 h, the infection rate was significantly reduced to 35.6 ± 3.5% (predicted mean difference 12.57, 95% CI 6.071–19.06, adjusted P = 0.0006), with the number of parasites per infected cell significantly increasing to 2.3 ± 0.5 (predicted mean difference 0.65, 95% CI 0.25–1.05, adjusted P = 0.0473) (Fig. 1). By 72 h, the infection significantly increased from 24 h (predicted mean difference −6.700, 95% CI −13.20 to −0.2043, adjusted P = 0.0431), reaching 42.3 ± 3.9%, and the mean parasite load was 2.4 ± 0.7 parasites per infected cell (Figs. 1 and 2B).Fig. 1Leishmania infantum intracellular parasite burden (A) and infection efficiency (B) at 4 (mean of parasites per cell: 2.1 ± 0.4, mean of infected cells: 48.2 ± 4.2), 24 (mean of parasites per cell: 2.3 ± 0.5, mean of infected cells: 35.6 ± 3.5), and 72 h (mean of parasites per cell: 2.4 ± 0.7, mean of infected cells: 42.3 ± 3.9) post-infection assessed microscopically in feline monocyte-derived macrophages. Asterisks indicate the statistical difference between timepoints (*P < 0.05, **P < 0.01). Adjusted P values are detailed in the text. Small dots represent individual replicatesFig. 2Feline monocyte-derived macrophages infected with Leishmania infantum at 4 h (A) and 72 h (B)

The cytokine expression profile of infected feline monocyte-derived macrophages is shown in Fig. 3 and illustrated in Fig. 4. Briefly, cytokine mRNA levels were quantified at each time point (i.e., 4, 24, and 72 h post-infection) and expressed as fold change relative to uninfected controls. IL-2 expression remained low at 4 h and 24 h (approximately 1–1.5-fold compared with controls) and significantly increased from 4 to 72 h (around onefold, predicted mean difference −0.9498, 95% CI −1.766 to −0.1336, adjusted P = 0.0185) and 24–72 h (around threefold, predicted mean difference −1.471, 95% CI −2.287 to 0.6544, adjusted P = 0.0001). IFN-γ mRNA levels were moderately elevated at 4 and 24 h (around 1.2-fold) and significantly higher at 72 h (around threefold, predicted mean difference −1.271, 95% CI −1.973 to −0.5683, adjusted P = 0.0001). TNF-α expression increased from 24 h (about 1.2-fold) to 72 h (≈ 2.5-fold, predicted mean difference −0.9841, 95% CI −1.687 to −0.2817, adjusted P = 0.0036). For IL-4 and IL-6, there were no significant differences between the three timepoints for these cytokines, remaining stable at all time points with only minor fluctuations (generally close to onefold compared with uninfected controls). IL-10 gene expression showed a significant decrease at 24 h (predicted mean difference 0.9188, 95% CI 0.1821 to 1.656, adjusted P = 0.0106), which persisted up to 72 h post-infection, with no statistically significant change between 24 and 72 h.Fig. 3. Relative mRNA gene expression (relative units) of cytokines IL-2, IFN-γ, TNF-α, IL-4, IL-6, and IL-10 from feline monocyte-derived macrophages infected by L. infantum at 4, 24, and 72 h in triplicate (standard deviations shown). Asterisks indicate the statistical difference between timepoints (*P < 0.05, **P < 0.01, ***P < 0.001). Adjusted P-values are detailed in the textFig. 4Cytokine expression in feline leishmaniasis. After macrophages phagocytize L. infantum promastigotes (in vivo after sand fly blood meal or in vitro after experimental infection of monocyte-derived macrophages), and after amastigote replication within the parasitophorous vacuole, the predominant response in the feline host is a Th1 cellular-protective one, with the expression of pro-inflammatory cytokines (IL-2, IFN-γ, and TNF-α), leading to a reduction in the parasitic load through reactive oxygen species (ROS)

Discussion

Feline monocyte-derived macrophages, when experimentally infected with L. infantum, showed a low and stable gene expression of the anti-inflammatory cytokines IL-4, IL-6, and IL-10, and upregulated expression of the pro-inflammatory cytokines IL-2, IFN-γ, and TNF-α. These findings indicate that, in experimental L. infantum infection, feline monocyte-derived macrophages show a predominance of pro-inflammatory cytokine expression, which may indicate early protective mechanisms. In contrast, in canine macrophages infected under similar in vitro conditions, L. infantum induced strong expression of IL-4 and IL-6 together with reduced IL-12 transcription, reflecting a Th2 immune response that is associated with disease progression [18]. Indeed, the Th2 humoral immune response with IL-4 and IL-10 cytokine expression is mostly associated with the formation of CICs and disease development. Likewise, IL-6 also acts as an anti-inflammatory mediator linked to acute-phase responses [18, 20, 29–31].

While it is tempting to assume that the pro-inflammatory cytokine expression pattern observed in this study could result in milder clinical signs, the absence of feline-specific studies precludes any definitive conclusions [32]. Nonetheless, the balance between lower anti-inflammatory cytokine expression and higher levels of pro-inflammatory cytokines demonstrated in our in vitro data may ultimately explain the milder clinical presentations often observed in Leishmania-infected feline hosts as compared with dogs. In particular, the clear upregulation of IL-2, IFN-γ, and TNF-α, particularly at 72 h, is synchronized with an increase in parasite load, indicating a potential correlation between the cellular immune response and L. infantum replication (Supplementary Fig. 1). Such a late cytokine upregulation may reflect a cell-mediated immune response similar to that described in field studies, where IFN-γ-producing cats were typically PCR-negative and clinically healthy [22]. This is confirmed by the high infection rates at 4 h paralleling low pro-inflammatory cytokines (e.g., IL-2, IFN-γ, and TNF-α), and a rise in the same cytokine level at 24 h, which eventually coincides with expanding intracellular parasite numbers.

A recent study also reported a Th1‑biased profile in asymptomatic cats, with significantly higher levels of IL‑12 and IFN‑γ compared with controls [24]. In fact, the rebound in infection rate at 72 h and peak cytokine expression imply that a pro-inflammatory environment might be related to higher parasite load, consistent with previous observations linking parasite burden and immunity [21, 22]. Indeed, IL-2 is known to support T-cell proliferation and maintenance of a pro-inflammatory environment, while IFN-γ promotes macrophage activation, and nitric oxide production is essential for intracellular parasite killing. Likewise, TNF-α synergizes with IFN-γ to enhance leishmanicidal activity and inflammatory cell recruitment [20]. To provide further insights into feline macrophages’ immune response and their potential role in controlling L. infantum infection, future studies could evaluate antigen-specific T-cell responses and their contribution to macrophage activation, as well as cytokine responses at the protein level and over extended timepoints. Furthermore, the study evaluated the cellular immune mechanisms in isolated cells from a healthy donor, thus erasing the background of the donor and minimizing external bias.

Conclusions

Overall, although these results should be interpreted as a initial experimental insight, this approach provided a better understanding of the immune mechanisms in feline leishmaniasis, suggesting the occurrence of an early and predominantly Th1-type pro-inflammatory response against L. infantum. The association between the rise in pro-inflammatory cytokine gene expression and the increase in parasite burden suggests an active, though only partially effective, Th1-mediated response, which may underlie the lower susceptibility and milder disease course typically observed in cats compared with dogs.

Supplementary Information

Additional file1 (TIFF 208 kb)