Changes in interferon-gamma and neopterin in female dogs undergoing ovariohysterectomy as elective spay or as treatment of pyometra

Abstract

The objective of this study was to determine serum concentrations of interferon-gamma (INF-γ) and neopterin (Np) in dogs with pyometra admitted for surgical treatment and to compare these concentrations to healthy dogs admitted for elective spay. The effects of the surgical procedure were also evaluated by measuring these markers in both groups of dogs before and after ovariohysterectomy. Our study indicates that pre-surgery concentrations of INF-γ (57.4 ± 26.0 pg/mL) and Np (5.6 ± 0.8 nmol/L) in healthy dogs were significantly lower compared to dogs with pyometra (124.3 ± 87.6 pg/mL for INF-γ; 7.0 ± 1.5 nmol/L for Np) (P < 0.05 in both cases). Furthermore, Np was lower in dogs with pyometra 3 days after surgery compared to healthy controls (P < 0.001). During the post-operative period, INF-γ showed no statistically significant changes in any of the groups, while Np showed lower serum concentration on day 3 than on day 0 in the pyometra group (P < 0.001). No statistically significant correlation was detected between serum concentrations of INF-γ and Np. These results indicate that pyometra causes alterations in serum concentrations of INF-γ and Np in female dogs compared to physiological levels before surgery and during the postoperative period.

Introduction

Interferon-gamma (INF-γ) is a type-II interferon, also called immunological interferon (1,2). The main sources of INF-γ are T-lymphocytes (2–4), natural killer (NK) cells, and natural killer T (NKT)-lymphocytes (5). More recently, its production by a family of immune cells called non-cytotoxic innate lymphoid cells (ILCs) has also been described (6,7). The production of INF-γ is induced predominantly by cytokines (8,9).

Interferon-gamma (INF-γ) plays a pivotal role in proper reactions of innate and adaptive immunity to viral infections and neoplasms, as well as to some bacterial and protozoan infections (1,7,10–13). Furthermore, since INF-γ presents immunoregulatory and antineoplastic activities, the usefulness of INF-γ determination for evaluation of the severity of inflammatory reactions has been demonstrated in neoplasms and bacterial diseases (10,12). Moreover, IFN-γ plays a central role in regulating the immune response after surgical trauma (14). It is an important activator of macrophages and stimulates the release of other cytokines, such as neopterin (Np) (15).

Neopterin (Np) is a non-specific, low-molecular mediator of the cellular immune response. It is considered an indicator of activation of macrophages and cell immune response (16) since Np is produced from guanosine-5′ triphosphate (GTP) in monocytes and macrophages activated during inflammation (17–19) and regulated by different cytokines. Besides INF-γ, other cytokines, such as tumor necrosis factor alpha (TNF-α), increase the release of Np by stimulating interleukin-4 (IL-4), and IL-10, while IL-12 inhibits the release of Np (20).

The main mechanism of Np action is modulation of oxidative stress (21). Being a mediator of the inflammatory response, Np is involved in generating active oxygen compounds (22). It is an important parameter that can be used for diagnosis and monitoring of treatment outcomes. The usefulness of its determination has been demonstrated in bacterial, viral, fungal, and protozoan infections, as well as in neoplasms and autoimmune diseases (19,23–25).

To assess the severity of the inflammatory process and to detect complications associated with inflammation after surgery, accurate monitoring is crucial. Before investigating whether inflammatory markers will serve to identify and grade systemic inflammation, normal concentrations in healthy animals need to be estimated. Similarly, before the effects of post-operative complications, such as wound infection, can be diagnosed, the normal inflammatory response to uneventful surgery has to be assessed.

Ovariohysterectomy (OHE) is one of the most frequent surgical procedures carried out in female dogs worldwide (26). This procedure is used both to prevent pet overpopulation (elective spay) and to treat pathologies of the uterus, such as pyometra. Pyometra is the most severe form of endometritis and one of the most common diseases of the reproductive tract in female dogs (27–29). It is also a potentially lethal bacterial infection of the uterus that is associated with systemic inflammation and affects on average 19% of all intact bitches (29). Determining the course of inflammatory processes is therefore of high importance.

The objective of the present study was to determine the INF-γ and Np concentrations in serum of dogs with pyometra admitted for surgical treatment and to compare these concentrations to healthy dogs admitted for elective spay. A further objective was to evaluate the effect of surgery by measuring the investigated variables before OHE and during the post-surgery period.

Materials and methods

The study was carried out in accordance with national animal protection regulations (Animal Experimentation Act, January 15, 2015) that are in agreement with European legislation on animal experiments. The authors also confirm that they have followed European Union (EU) standards for the protection of animals used for scientific purposes.

A total of 40 dogs took part in the study. The dogs were admitted to the Department and Clinic of Animal Reproduction, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Poland from April to October 2018 for an ovariohysterectomy, either as treatment for pyrometra or for spaying.

The study group consisted of 30 bitches with pyrometra from 4 to 12 y of age and with body weight (BW) ranging from 12 to 50 kg. The dogs included 6 breeds (Rottweiler, German shepherd, golden retriever, boxer, dachshund, and cocker spaniel) as well as mixed-breeds, with a body condition score (BCS) of 2 to 4/5. All dogs were examined clinically and additional laboratory tests (hematological, biochemical) were carried out, leading up to a preliminary diagnosis of pyometra and surgical treatment (ovariohysterectomy). The diagnosis of pyometra was confirmed by a post-operative histopathological examination of the uterus and ovaries at the Department of Pathological Anatomy, University of Life Sciences. Uterine pus from all dogs with pyometra was submitted for bacteriology. In addition, the bacteriological analyses were conducted in the laboratory of the Department and Clinic of Animal Reproduction, University of Life Sciences and Escherichia coli was isolated from the uterine content samples.

The control group consisted of 10 clinically healthy bitches from 4 to 10 y of age and with body weight ranging from 6 to 42 kg. The dogs consisted of 4 different breeds (German shepherd, golden retriever, boxer, and dachshund) as well as mixed breeds, with a BCS of 2 to 4/5. The dogs were admitted for elective OHE (spay). The healthy control dogs were all in anestrus as determined by history data and clinical and additional examination findings. Blood samples were collected from vena cephalica into silicone tubes with clot activator (Vacutest Kima, Piove di Sacco PD, Italy) before surgery (D0), and 3 d (D3) and 10 d (D10) after surgery. The samples were centrifuged at 3000 × g for 15 min and supernatant was transferred to Eppendorf tubes and frozen at −80°C until analysis.

Interferon-gamma (IFN-γ) was determined using the commercial enzyme-linked immunosorbent assay (ELISA) test (Canine IFN-gamma Quantikine ELISA Kit; R&D Systems, Minneapolis Minnesota, USA). Neopterin (Np) was assayed using a commercial ELISA kit (Neopterin ELISA Kit; Immuno-Biological Laboratories, Hamburg, Germany) (30). The absorbance was read with an ELISA — LAP-SYSTEM reader, at wavelengths of 450 nm and 630 nm as reference (ELx800, BioTek Intruments, Winooski, Vermont, USA). The 2 assays showed adequate precision (coefficient of variation < 15%) and accuracy determined by linearity under dilution (coefficient of correlation close to 1).

The distribution of the studied variables was checked by using the Kolmogorov-Smirnov test. Values of the analyzed parameters measured on the quotient scale were characterized by means of mean and standard deviation or median and the range of variation. Values below assay range were replaced by half of the lower value of assay range for statistical analyses. The analysis of variance (ANOVA) with repeated measurements or the non-parametric Friedman ANOVA test were used to determine changes over time, for a single classification, while the ANOVA test with repeated measurements divided into groups was used to assess the differences between the groups. Pearson’s correlation was done to evaluate possible correlation between serum concentrations of INF-γ and Np. In all cases, P < 0.05 was considered significant for 2-sided analyses.

Results

There were no statistically significant differences between groups in terms of age, body weight, and BCS (P > 0.05).

Serum concentrations of INF-γ in both groups of dogs at different time points are shown in Figure 1. Before surgery, the mean (± SD) INF-γ value in the group of dogs with pyometra (124.3 ± 87.6 pg/mL) was higher than in the group of healthy dogs (57.9 ± 26.4 pg/mL) (P < 0.05). Interferon-gamma (INF-γ) showed no statistically significant differences between groups on days 3 and 10 after surgery or within groups during the study period.

Figure 1.

Serum concentration of interferon-gamma in healthy control dogs (blue squares) and dogs with pyometra (red circles) before and 3 d (D3) and 10 d (D10) after ovariohysterectomy.

^* = P < 0.05 between groups.

Changes in serum concentrations of Np in dogs of the 2 groups at different time points are shown in Figure 2. The data demonstrate that the mean Np concentration in the group of dogs with pyometra was significantly higher before surgery and significantly lower on day 3 after surgery than the Np concentration in healthy dogs (P < 0.001, in both cases), while no significant differences were detected between the groups on day 10. The intragroup comparative analysis revealed that Np concentration did not vary significantly in control dogs, while in dogs with pyometra, serum Np significantly decreased on day 3 after surgery (mean ± SD; 4.0 ± 0.7 nmol/L) compared to levels before surgery (7.0 ± 1.5 nmol/L) (P < 0.001). No statistically significant correlation was detected between serum concentrations of INF-γ and Np.

Figure 2.

Serum concentration of neopterin in healthy control dogs (blue squares) and dogs with pyometra (red circles) before and 3 d (D3) and 10 d (D10) after ovariohysterectomy.

^*** = P < 0.001 between groups.

^# = P < 0.001 versus before withing group.

Discussion

Our study findings demonstrated that the mean values of the markers of cell-mediated, non-specific immunity were higher before surgery in the dogs with pyometra than in those in healthy dogs. Pyometra has been associated with endotoxemia and bacteremia, which could be one of the main reasons that numerous cell-mediated serum biomarkers are improperly synthesized (31). Higher levels of INF-γ and Np in affected dogs could therefore have resulted from the ongoing infection and/or purulent inflammation in the uterus, although the effect of sex hormones could have influenced the levels and the difference between the groups. Nevertheless, the results of the present study are in accordance with previously reported data in humans (10,32).

It is well-known that during the inflammatory processes monocytes and macrophages are activated, which are considered the inducers of Np synthesis in blood (directly affected by IFN-γ). Furthermore, it is known that these 2 cytokines are highly interrelated (18,33). As markers of macrophage activation and cell-mediated immune response (15,17), Np and INF-γ are the essential parameters for the diagnosis of pyometra and their determination is useful for assessing the intensity of the inflammatory reaction during pyometra. Our results are consistent with those reported by Kostro et al (23), who have demonstrated that determination of Np is particularly useful for monitoring and prognosis of mastitis-metritisagalactia (MMA) in sows. If high values of serum Np in sows persist during the therapy, the treatment is considered ineffective.

Dorresteijn et al (34) reported that the systemic administration of endotoxin to healthy volunteers caused an increase in IFN-γ serum levels. The inflammatory process that was noticed in the dogs with pyometra was also confirmed by the results for C-reactive protein (CRP) and haptoglobin (Hp), which were determined in parallel in our study as the traditional biomarkers of the intensity of inflammation (27,35).

Efficient mechanisms of cell-mediated immune responses are also one of the basic elements that determine an uneventful post-operative period in dogs after ovariohysterectomy. When post-operative complications develop, dysfunction of monocytes/macrophages leads to an inability to fight the infection-induced inflammatory response, which ultimately results in prolonged recovery or even death. According to our results, the lowest serum concentrations of Np and IFN-γ were observed on day 3 after surgery in dogs with pyometra. This shows additionally transient immunosuppression resulting from the development of the additional inflammatory response caused by surgery, which is the body’s defence against further degradation of the damaged tissues. Neopterin (Np) and IFN-γ can be considered as acting proteins, which are induced by the same mechanisms (18,36).

There are, however, different mechanisms for stimulating IFN-γ synthesis, which have different effects depending on the causative event, i.e., surgical procedure, sepsis, endotoxemia (14,37,38). In our study, serum concentrations of Np in dogs with pyometra already significantly decrease several hours after surgical trauma. This results from the additional activation of immune cells (monocytes/macrophages) due to tissue discontinuity and the development of the local inflammatory process. Moreover, the decrease in concentration of IFN-γ after surgery in dogs with pyometra, although not significant, is discussed as a cause of post-operative immunosuppression that predisposes to post-operative infectious complications (14). Because IFN-γ synthesis is reduced post-operatively due to a lack of IL-12, there is no further stimulation of monocyte and macrophage activity (37). Our results are similar to those presented by Holl et al (39) who reported lower Np concentration during the 24-hour period before induction, with a slower recovery to baseline level occurring during the 96-hour time period after surgery. Moreover, Franke et al (14) noticed significantly decreased levels of INF-γ after cardiac surgery in humans.

In our study, the mean serum values of Np and IFN-γ were found to have increased on day 10 after surgery and were in similar concentrations as before surgery in both study groups. This shows that normal biological functions of the cell-mediated immune response were restored due to gradual subsidence of the local inflammatory response caused by the ovariohysterectomy. Moreover, in the case of the dogs with pyometra, it confirms that the inflammatory response in the genital tract caused by pyometra was eliminated. This was confirmed by normalization of acute-phase proteins (APPs) on day 10 after surgery, compared to baseline values in both groups of dogs (27,35). Similar results were reported by Wołoszczuk et al (40) in humans, describing the dynamics of changes in Np and IFN-γ after surgical procedures.

This study has some limitations. Ideally, C-reactive protein (CRP) and haptoglobin (Hp) should have been determined in the present study in order to assess the possible relationship between these acute-phase proteins that are highly studied in different inflammatory pathologies, including pyometra and Np and IFN-γ. Furthermore, the possible relationship between biomarkers studied in the present work and the severity of pyometra and/or the presence of endotoxemia and sepsis should have been determined in order to assess the possible reason for high inter-individual variability in concentrations of both Np and IFN-γ before surgery in dogs with pyometra. Additional studies are therefore required to further investigate the behavior of Np and IFN-γ in inflammation in relation to other pro- and anti-inflammatory biomarkers and the severity of the pathology.

In conclusion, the results of the present study revealed that pyometra causes alterations in serum concentrations of interferon-gamma (INF-γ) and neopterin (Np), markers of cell-mediated, nonspecific immunity, in female dogs. These data suggest that these parameters might be useful for pyometra diagnosis and monitoring of treatment outcome.