Progesterone Alters Kynurenine Pathway Activation in IFN-γ-Activated Macrophages - Relevance for Neuroinflammatory Diseases.

We have previously demonstrated that the kynurenine pathway (KP), the major biochemical pathway for tryptophan metabolism, is dysregulated in many inflammatory disorders that are often associated with sexual dimorphisms. We aimed to identify a potential functional interaction between the KP and gonadal hormones. We have treated primary human macrophages with progesterone in the presence and absence of inflammatory cytokine interferon-gamma (interferon-γ) that is known to be a potent inducer of regulating the KP enzyme. We found that progesterone attenuates interferon-γ-induced KP activity, decreases the levels of the excitotoxin quinolinic acid, and increases the neuroprotective kynurenic acid levels. We also showed that progesterone was able to reduce the inflammatory marker neopterin. These results may shed light on the gender disparity in response to inflammation.

Introduction

KP, neuroinflammation, and psychiatric disease

The KP is the major catabolic pathway for tryptophan (TRP) and is known to be altered in a number of neurodegenerative diseases, including amyotrophic lateral sclerosis,1 Parkinson’s disease,2 and Alzheimer’s disease.3 Neurodegenerative diseases are associated with chronic inflammation.4 The KP has been shown to be upregulated by different inflammatory mediators such as proinflammatory cytokines, interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α).5,6 Our group has previously shown that the KP is activated by IFN-γ in human macrophages.7 The KP is also known to be strongly altered in a number of neuropsychiatric disorders, including schizophrenia,8,9 depression,10 and autism.11 These disorders are also associated with chronic, and possibly causative, neuroinflammatory features.12,13 In human beings, the levels of cytokine interleukin-6 have been found to be significantly higher in men than in women of the same age.14 Recent molecular data of primary CD4+ T cells support a sexually dimorphic immune response in human beings.15 In this study, gender was the most significant attributable source of genetic variation for the immune response. While the interplay between gonadal hormones and the immune system is likely to be a key factor in gender disparity for neurodegenerative and psychiatric disorders,16 the mechanisms for this complex interplay are still unclear.

Gender and pathology

Gender disparity in onset and progression is observable in most neurodegenerative and psychiatric disorders that are all associated with neuroinflammation.17 While mechanisms are still unclear, evidence is mounting that the immune system is influenced by sex hormones. Bini et al showed increased immune activity and survival in female and castrated male mice in a tuberculosis challenge experiment. Survival and immune activity were immediately improved after gonadectomy in males.18 Another study showed an increase in IFN-γ and TNF-α expression with progesterone treatment in female mice and a decrease in testosterone in infected males. Infection and subsequent inflammation have been shown to induce expression of the progesterone receptor in mice, further compounding evidence for a complex interplay between sex hormones and the immune response in animal models.19 Research on associations between progesterone levels and cognition in schizophrenia is sparse and with mixed results. However, progesterone has been shown to have some neuroprotective benefits.20 Progesterone actions contribute to positive effects on cognition, sensory motor performance, and other aspects of neuropsychiatric illnesses,21 suggesting possible ways in which progesterone may beneficially affect symptomatology in female patients. There are no published studies evaluating the benefit of progesterone as a treatment for schizophrenia.

We hypothesize that the KP provides a mechanistic framework in which the immune system and gonadal hormones interact and affect the health of central nervous system. We expect gonadal hormones to alter the KP metabolism of immune cells, their microenvironment, and their cellular interactions.

Materials and Methods

Cultures of human monocyte-derived macrophages

This research complied with the principles of the Declaration of Helsinki. Subjects gave their written, informed consent to participate in the research, which was approved by the Macquarie University Human Research Ethics Committee. Briefly, human peripheral blood mononuclear cells were isolated from 100 mL of blood from healthy volunteers using a standard Ficoll-Paque (Pharmacia) density separation method.7,22 Monocyte-derived macrophages were obtained using a classic adherence method. After eight days, the classic medium was replaced by AIM-V, a serum-free medium (Life Technologies) to limit any aspecific activation. After 11 days, 98% of the adherent cells expressed macrophage markers, including CD68+++, CD11c++, and CD16+.23

Treatment

Progesterone stock was made from progesterone (Sigma-Aldrich Co.) and reconstituted in 100% ethanol before being diluted in a cell culture medium with a final concentration of <0.02% (v/v) of ethanol using a stock concentration of 2 mg/mL at 100 nM, 10 μM, and 100 μM, and then incubated for 48 hours at 37 °C in a humidified atmosphere containing 5% CO2.

Quantification of the KP metabolites

As previously optimized, production of the KP metabolites was quantified at 48 hours.11 We used ultra-high performance liquid chromatography to quantify TRP, kynurenine (KYN), and kynurenic acid (KYNA) and gas chromatography–mass spectrometry to quantify quinolinic acid (QUIN) and picolinic acid (PIC).24

Statistics

IBM SPSS statistics software version 21 was used for the analysis (IBM Corporation). Owing to the heteroscedastic nature of the metabolite data, raw values were log transformed to achieve normal distribution for analysis. One-way analysis of variance was used to assess the differences in the treatment groups, with post hoc Bonferroni tests for differences between treatment and incubation time groups.

Results

We found progesterone attenuates IFN-γ-induced KP activation and alters IFN-γ-induced metabolism in human macrophages. As previously reported,7 KYN/TRP ratio increases in the culture supernatant of IFN-γ-treated human macrophages. We found that an attenuation of this KYN/TRP ratio increased following treatment with 100 μM progesterone (P × 0.001; Fig. 2D). In these IFN-γ-stimulated cells, treatment with progesterone significantly increased KYNA levels at 100 nM (P < 0.001), 10 μM (P × 0.043), and 100 μM (P × 0.003; Fig. 1C) and increased TRP levels at 100 μM (P < 0.001). KYNA/QUIN is significantly increased at 100 nM (P < 0.001) and 10 μM (P × 0.003) but not at 100 μM (P × 0.483; Fig. 2E). PIC/QUIN ratio is increased at 100 μM (P × 0.001; Fig. 2F).

Figure 2

Effects of 100 nM, 10 μM, and 100 μM progesterone on primary human macrophages in the absence and presence of IFN-γ: (A, D) KP activity as described by KYN/TRP ratio, (B, E) KYNA/QUIN ratio, and (C, F) PIC/QUIN ratio. Error bars denote standard deviation (n × 3).

Figure 1

Effects of 100 nM, 10 μM, and 100 μM progesterone on primary human macrophages in the absence and presence of IFN-γ: (A) TRP production, (B) KYN production, (C) production of KYNA, (D) production of anthranilic acid, (E) production of PIC, and (F) production of QUIN. Error bars denote standard deviation (n × 3).

In unstimulated cells, progesterone treatment for 48 hours decreased PIC levels at 10 μM (P × 0.014) and 100 μM (P × 0.002; Fig. 1E), decreased QUIN levels at 100 μM (P < 0.001; Fig. 1F), increased KYNA/QUIN ratio at 100 nM (P × 0.026; Fig. 2B), and increased PIC/QUIN ratio at 100 μM (P < 0.001; Fig. 2C). Treatment of unstimulated cells with 10 μM and 100 μM of progesterone for 48 hours decreased neopterin levels (P × 0.001, P × 0.007; Fig. 3A), and combined treatment with IFN-γ and 100 μM progesterone decreased neopterin levels (P < 0.001; Fig. 3B).

Figure 3

Effects of 100 nM, 10 μM, and 100 μM progesterone on neopterin production in primary human macrophages: (A) in the absence of IFN-γ and (B) in the presence of IFN-γ. Error bars denote standard deviation (n × 3).

Discussion

We showed that progesterone is able to influence the production of both neopterin and KP metabolites. Neopterin is a well-known marker of immune system activation.25 The dose-dependent reduction of neopterin levels following progesterone treatment indicates an anti-inflammatory effect. This effect persists in combined treatment with IFN-γ and progesterone but seems to be present only when treated with 100 μM of progesterone. This is a much higher concentration of progesterone than would occur under normal physiological conditions. However, high progesterone doses are prescribed to women in “at-risk” pregnancies, which could likely cause localized concentrations to approach 100 μM.

We also found that progesterone can significantly influence the production of several KP metabolites. We previously described that the levels of KYN/TRP increase in response to IFN-γ stimulation in macrophages.7 We observed a significant attenuation of this increase by treatment with 100 μM of progesterone (P < 0.001). This is likely to be associated with an anti-inflammatory effect of progesterone that has been described previously.26,27

Progesterone increases the production of the immunomodulatory KYN and the neuroprotective KYNA and decreases biosynthesis of the excitotoxin and proinflammatory QUIN by IFN-γ-induced macrophages (Fig. 2). KYN is known for its immunomodulatory ability, especially during pregnancy.28,29 Both KYNA and KYN directly activate the aryl hydrocarbon receptor, a receptor known to have both pro- and anti-inflammatory effects.30 KP modulation by progesterone could represent a mechanism for immune regulation. Interestingly, KYNA is also an alpha-7 nicotinic acetylcholine receptor (α7nAChr) antagonist. This receptor seems to play a crucial role in cholinergic regulation of macrophage activity.31 Antagonism of the α7nAChr is associated with suppression of the anti-inflammatory effect of vagal nerve signaling, generally producing a net pro-inflammatory effect. Additionally, a depletion of TRP by increased KP activity may affect serotonin and subsequent melatonin production. Melatonin is also an α7nAChr agonist.32 A reduction in an α7nAChr agonist would be expected to reduce anti-inflammatory effect on macrophages.33 In this study, inflammatory marker neopterin is reduced with increased KP activity and the resulting KYNA production. The nature of the study (in vitro) and the subsequent lack of nicotinic or vagal stimulation may possibly account for this effect. With no anti-inflammatory signaling to be suppressed and additional immunomodulation coming from increased KYN, the overall pattern is one of decreased immune activation. This pattern may well shift in in vivo conditions.

The potentiation of KYNA production by progesterone does not appear to be dose dependent. The increase in KYNA and decrease in QUIN in IFN-γ-stimulated and progesterone-treated samples resulted in an overall increase in KYNA/QUIN ratio. KYNA is an N-methyl-d-aspartate-receptor (NMDAR) antagonist34 and is usually considered as a neuroprotective molecule,35 whereas QUIN is an NMDAR agonist and a well-known neurotoxin, gliotoxin, and pro-inflammatory molecule.36 The overall effects of progesterone can be considered as beneficial, resulting in a decrease in deleterious QUIN together with an increase in neuroprotective KYNA and a decrease in the inflammation marker neopterin. It is important to mention that, among all the human cell types, activated macrophages are able to produce the highest levels of QUIN, but produce far less KYNA.

These results may have a specific relevance to schizophrenia. It has become increasingly evident that inflammation is involved in the development of schizophrenia.37 This anti-inflammatory role of progesterone may explain at least, in part, why schizophrenia develops differently in females. A shift in KYNA/QUIN ratio has been previously reported in schizophrenia, with a reduction in QUIN38 and an increase in cerebrospinal fluid KYNA.39 The relationship of progesterone levels to clinical schizophrenic presentation and treatment response in either gender is still unclear due to limited and conflicting data. It is possible that progesterone, via its action on the KP, could play a key role in the development, symptomology, and severity of schizophrenia.

Our findings on treatment with progesterone support our hypothesis that gender disparity in diseases may be due, at least in part, to hormonal effects on the KP and inflammation.