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Plasma Inammatory Cytokines and Depressed Patients With Comorbid Pain: Improvement by Ketamine

Background: Depression and pain frequently coexist clinically. Ketamine has analgesic and antidepressant effects, but few studies have evaluated individual differences in antidepressant outcomes to repeated ketamine in depressed patients with comorbid pain. Our aims were to determine the difference in ketamine’s antidepressant effects in depressed patients with or without pain and then to examine whether inammatory cytokines might contribute to ketamine’s effect. Methods: Seventy-eight patients with major depressive disorder received six infusions of ketamine. Plasma levels of 19 inammatory cytokines were assessed at baseline and post-infusion (day 13 and day 26) using the Luminex assay. Plasma inammatory cytokines of sixty healthy controls (HCs) were also examined. Results: At baseline, the levels of GM-CSF, IL-1β and IL-6 were higher in pain group than in non-pain and HC groups. Pain group had better antidepressant outcomes than non-pain group. Pain group showed a greater decrease in IL-6 at day 13 and a greater decrease in IL-10, MIP-3α, IL-1β, IL-5 and IL-6 at day 26 than non-pain group. In the pain group, the changes in IL-6 levels were associated with improvement in pain intensity (β=0.347, t=2.159, P=0.038) and depressive symptoms (β=0.590, t=4.201, P<0.001) at day 13. The Sobel test showed indirect effects between decreases in IL-6 levels and improvement in depressive symptoms (Z=2.026, P=0.043). Conclusion: This study suggested that an elevated inammatory response plays a key role in individual differences in depressed patients with or without pain. Ketamine showed great antidepressant and analgesic effects in depressed patients with pain, which may be related to its anti-inammatory effect.

exhibit depressive-like behavior and, conversely, in rodent models of depression that also display altered nociceptive responses [11][12][13]. Imbalanced peripheral proin ammatory cytokine levels have also been independently observed in patients with depression and pain and in comorbid patients compared to healthy controls. For example, increased IL-6 levels can be found in patients with chronic pain and comorbid depressed mood, and IL-6 levels and depressive symptoms were positively associated with pain intensity [9]. TNF-α is another general in ammatory mediator reported to be increased in patients with depression and comorbid with pain, and augmented peripheral levels of TNF-α were associated with reduced pain thresholds in a correlative analysis [14]. Elevated peripheral C-reactive protein levels and depression symptoms were observed in patients with bromyalgia or sciatica, while higher C-reactive protein levels correlated with greater depressive symptoms [15,16]. Thus, taken together, these data suggest that a persistent in ammatory response may underlie comorbid depression and pain, or at least partly contribute to the development of this comorbidity.
Currently, some analgesics and antidepressants are being used to treat this comorbidity; however, sometimes, there is limited clinical remission [17,18]. Ketamine is an N-methyl-D-aspartic acid receptor antagonist with anesthetic and analgesic effects and has been used for acute pain for several decades.
Recently, an increasing number of clinical studies have identi ed that a single subanesthetic dose of ketamine has a fast and robust antidepressant effect in patients with treatment-resistant depression [19,20]. Our previous results also showed greater effectiveness and longer remission periods with six infusions of intravenous ketamine for patients with major depressive disorder (MDD) [21], consistent with other research [22]. Ketamine infusions also signi cantly reduced pain and depression in patients experiencing refractory neuropathic pain syndromes and depressive comorbidities [23,24] and even successfully relieved depressive symptoms, suicidal ideation and neuropathic pain in an adolescent with severe depression, suicidality, and neuropathic leg pain who failed multiple antidepressant and analgesic modalities [25]. In addition, oral ketamine thrice daily for six weeks was proven to have superior antidepressant effects compared to diclofenac for the treatment of depressed patients suffering from chronic pain [26].
Based on evidence from previous studies, ketamine may be ideal for the treatment of comorbid pain and depression, however, few clinical studies have evaluated individual differences in antidepressant outcomes to repeated ketamine infusions in patients with MDD and comorbid pain. Moreover, although plasma levels of in ammatory cytokines decreased after six infusions of ketamine administration based on our previous results [27], the roles of cytokines in ketamine's effect on concurrent pain and depressive symptoms have not been explored. In the present study, we aimed to rst determine differences in ketamine's antidepressant effects in depressed patients with and without the presence of painful symptoms and then to examine whether cytokines might contribute to ketamine's effect in depressive patients with the presence of painful symptoms.

Participants
We present a post hoc analysis of an original study designed to assess the antidepressant response of six adjunctive ketamine infusions in patients with MDD [21,28]. This study was approved by the Clinical Research Ethics Committee of the A liated Brain Hospital of Guangzhou Medical University. All participants provided informed consent prior to participation.
This study included 78 patients with MDD who received six doses of ketamine, along with 60 healthy controls (HCs) matched with patients for age and sex. The main inclusion criteria for MDD patients were as follows: diagnosis of MDD established using the Diagnostic and Statistical Manual of Mental Disorders (DSM-V) criteria; age between 18 and 65 years; 17-item Hamilton Depression Rating Scale (HAMD-17) score ≥ 17 at screening without hallucination or delusion; and treatment resistance de ned as the failure of two adequate antidepressant trials or a suicidal tendency con rmed by a Beck Scale for Suicide Ideation-part I score ≥ 2 at screening. The exclusion criteria included the presence of alcohol or substance dependence or any serious or unstable medical conditions, including neurological, endocrine, rheumatic and infective diseases. Patients taking anti-in ammatory agents were also excluded. Current psychotropic medication had to be stable for ≥ 4 weeks, and the same dose was maintained during the six-infusion period. Additional detailed information regarding these participants has been described in our previous studies [21,28,29].

Study design
The patients received ketamine three times weekly for two weeks. The detailed study design and methods have been previously published [21,28,29]. Intravenous ketamine (0.5 mg/kg) was administered over 40 minutes following an overnight fast via IV intravenous pump continuous infusion.
Depressive symptoms were assessed using the Montgomery-Asberg depression rating scale (MADRS) by clinicians at the pretreatment baseline, 24 h after each infusion and again 14 days after the 6th infusion (day 26). A response was conventionally de ned as a 50% or more reduction from baseline in MADRS score at 24 h after the 6th infusion (day 13). Remission was de ned as a MADRS total score ≤ 10 at day 13.
Pain intensity was measured using the short-form McGill Pain Questionnaire (SF-MPQ). The main component of the SF-MPQ consists of the sensory index, affective index, present pain intensity (PPI) index and visual analog scale (VAS). The VAS was assessed at the same timepoint as the MADRS, but the sensory index, affective index and PPT were assessed at baseline, day 13 and day 26. Based on the presence or absence of pain using the SF-MPQ, 36 (46.2%) MDD patients had pain symptoms at the baseline.

In ammatory cytokine measurements
The patients provided blood samples at baseline, day 13 and day 26, and HCs provided only a single blood sample. Blood samples were collected into EDTA tubes between 8:00 and 10:00 AM after an overnight fast. Tubes were immediately stored at + 4°C and then centrifuged (3000 rpm/min at + 4°C) for 10 minutes within 1 hour. Plasma was obtained and stored at − 80°C.

Statistical analysis
Of the 78 MDD patients included, 7(9.0%) lacked blood sample at day 26, four of them were in non-pain group and three in pain group. Thus, these data were analyzed based on the intent-to-treat with expectation maximization algorithm interpolation method.
First, baseline demographic variables and clinical symptoms between groups (pain vs. non-pain) were statistically evaluated using Student's t-test for continuous variables with a normal distribution and chisquare test for categorical variables.
Next, the group comparisons of baseline cytokine levels among the pain group, non-pain group and HC group were examined using multivariate analysis of covariance (MANCOVA), with age, sex and body mass index (BMI) as covariates. Post hoc analysis was used to compare the differences within each group. Cohen's d was calculated to measure their difference. Prior to the analyses, data for in ammatory cytokines were natural log-transformed.
Then, changes in MADRS scores, VAS, sensory index, affective index and PPT over time and group differences were assessed using linear mixed models with group (pain vs. non-pain) and time (baseline, 24 h after each infusion or day 13, day 26) as factors. Baseline demographic and clinical variables that differed between groups were entered as covariates in linear mixed models. Bonferroni-corrected post hoc comparisons were used to calculate the group differences at each follow-up point.
Based on our previous study, the concentrations of several cytokines, including GM-CSF, fractalkine, IFN-γ, IL-10, IL-12p70, IL-17A, IL-1β, IL-2, IL-4, IL-23, IL-5, IL-6, IL-7 and TNF-α, were downregulated after six ketamine infusions in patients with MDD regardless of the presence of comorbid pain, so we further compared the changes in cytokine levels from baseline to follow-up points (day 13 and day 26) between the two groups using analysis of covariance, with their baseline levels as covariates.
Finally, linear regression analyses were used to further test whether changes in depressive symptoms individually correlated with changes in in ammatory cytokine levels in the pain group and the non-pain group and whether changes in pain symptoms correlated with changes in in ammatory cytokine levels in the pain group. Mediation analyses using the Sobel test (http://www.quantpsy.org/sobel/sobel.htm) were performed to assess whether the relationships between changes in in ammatory cytokine levels and changes in MADRS scores were mediated by changes in VAS scores in the pain group.
All statistical analyses were performed using IBM SPSS Statistics version 22, and p-values < 0.05 were considered statistically signi cant. P-values were adjusted for multiple comparisons using false discovery rate correction.

Demographics
At baseline, BMI and years of education in the pain group were signi cantly greater than in the non-pain group; other baseline demographic or clinical characteristics showed no statistically signi cant differences between groups. The patients with pain showed a signi cantly shorter time to an antidepressant response (F = 2.122, P = 0.036) and to depression remission (F = 2.172, P = 0.032) and had a higher response rate (χ 2 = 6.026, P = 0.014) and remission rate (χ 2 = 4.373, P = 0.037) than the patients without pain.
MADRS scores at baseline did not differ between the pain group and the non-pain group. The linear mixed model with MADRS scores showed a signi cant group main effect (F = 8.066, P = 0.006), time main effect (F = 86.986, P < 0.001) and group-by-time interaction (F = 3.461, P = 0.001). Signi cant reductions in MADRS scores were found at 24 h after the rst infusion compared to baseline scores, and these reductions were maintained over the subsequent infusion period as well as on day 26 in both groups (all P < 0.05, Fig. 2 Table 2). The pain group had lower MADRS scores than the non-pain group from the 2nd infusion to the 6th infusion and on day 26 (all P < 0.05). The largest signi cant differences in MADRS scores between the pain group and the non-pain group were observed at the 6th infusion (Cohen's d = 0.565).

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The VAS scores, sensory index, affective index and PPI in the pain group at baseline are summarized in Table 1. The linear mixed model showed a signi cant group main effect, time main effect and group-bytime interaction with VAS scores (group: F = 57.124, P < 0.001; time: F = 390.614, P < 0.001; interaction: F = 63.651, P < 0.001), sensory index (group: F = 45.354, p < 0.001; time: F = 33.782, P < 0.001; interaction: F = 48.499, P < 0.001), affective index (group: F = 71.426, P < 0.001; time: F = 46.391, P < 0.001; interaction: F = 64.352, P < 0.001) and PPI (group: F = 77.267, P < 0.001; time: F = 35.039, P < 0.001; interaction: F = 62.254, P < 0.001). In the pain group, large and signi cant reductions in VAS scores were found at 24 h after the rst infusion compared to baseline scores and were maintained over the subsequent infusion period as well as on day 26 (all P < 0.05), and signi cant reductions in sensory index, affective index and PPI were found at the 6th infusion and on day 26 compared to baseline scores (all P < 0.05, Fig. 2, 3 and Supplementary Table 2). Signi cant differences between the pain group and the non-pain group in VAS scores were found during the preceding ve infusions and day 26. No signi cant differences between groups in sensory index, affective index or PPI were observed at day 13 and day 26 (all P > 0.05) .

Differences in cytokine changes after ketamine treatment
Based on the mean change in the levels of in ammatory cytokines, most of them decreased after ketamine treatment ( Fig. 4 and Supplementary Table 3). The pain group showed a signi cantly greater decrease in IL-6 levels on day 13 and a greater decrease in IL-10, MIP-3α, IL-1β, IL-5 and IL-6 levels on day 26 than the non-pain group (all P < 0.05).

Clinical effects and in ammatory cytokine alterations after ketamine infusions
In the non-pain group, linear regression analyse showed that none of the cytokine alterations were associated with the reduction in MADRS scores following ketamine on day 13 or day 26 (all P > 0.05). In the pain group, the changes in IL-6 levels were

Discussion
Three important ndings of the present study are as follows: First, the depressed patients with comorbid pain had an elevated in ammatory response compared with the depressed patients without pain and healthy controls. Second, repeated subanesthetic doses of ketamine had signi cantly superior antidepressant effects in the depressed patients with comorbid pain compared with the patients without pain. Third, ketamine exerted greater downregulation effects in the depressed patients with pain than in the patients without pain. This study is the rst to examine the role of plasma in ammatory cytokines in clinical individual differences in depression comorbid with pain. In addition, this is the rst study to explore clinical individual differences in ketamine's antidepressant effects in individuals with comorbid depression and pain, as well as the role of plasma in ammatory cytokines in ketamine's antidepressant response in those patients.
The present study showed that 46.2% of patients with MDD had comorbid pain, consistent with previous reports that the comorbidity rate of chronic pain and depression is approximately 40-60% [3,4]. Our ndings that depressed patients with or without pain showed similar severity of depressive symptoms suggested that painful symptoms were independent of the degree of depression, which was inconsistent with previous results that patients with MDD comorbid with chronic pain suffered from more severe depression [30,31].
A wealth of evidence supports the hypothesis that excessive activation of in ammation contributes to the pathophysiology of the comorbidity of pain and depression. Microglial activation in the hippocampus and thalamus was found in patients suffering from chronic fatigue syndrome who exhibited pain and depression using positron emission tomography scans [32], and microglial activation and increased in ammatory cytokine expression were found in pain-and mood-related brain regions in rodent models of depression-pain comorbidity [13,33]. Activation of the in ammatory response was also found in comorbid depression and pain patients. For example, higher plasma IL-6 levels were found in patients with chronic back pain and comorbid depression and in patients with burning mouth syndrome and depressive symptoms than in healthy controls [9,10]. In addition, higher serum IL-6 levels in patients suffering from burning mouth syndrome and depressive symptoms exhibited more pain [9]. In the present study, in addition to IL-6, other in ammatory cytokines, including GM-CSF, fractalkine, IL-10, MIP-3α, IL-13, IL-17α, IL-1β, IL-2, IL-6 and MIP-1β, were elevated in depressed patients with pain, while the levels of IL-4 decreased. Furthermore, plasma levels of GMCSF, IL-1β and IL-6 in depressed patients with pain were higher than those in patients without pain. An excessive in ammatory response may contribute to individual differences in risks for the comorbidity of pain and depression. A preclinical study reported increased levels of the in ammatory cytokines IL-6, IL-1β, TNF-α, IL-4 and IL-10 in spared nerve ligation rats with a depression-like phenotype but not rats without a depression-like phenotype [34]. Polymorphisms related to the in ammatory response may be moderators of depressive reactions to stress. IL-1β genetic variation and attendant increased IL-1β expression were found to be associated with high risks for stress-induced depression in a large cohort of youths [34]. Thus, it is likely that genetic variants that enhance immune reactivity might create vulnerability to pain and depression comorbidity. Further studies examining differences in immune gene expression between depressed patients with or without pain are needed to con rm this speculation.
Pain adversely affects the treatment response and prognosis of depression and vice versa. Patients with comorbid pain and depression were reported to experience a worse response to analgesic therapy than those without depressive symptoms [35]. Patients who had more severe pain symptoms prior to selective serotonin reuptake inhibitor treatment experienced poorer responses [30]. For depressed patients with pain in our study, the rate of response to six infusions of ketamine was 73.1%, and the remission rate was 48.1%, which were signi cantly higher than those without pain. Moreover, patients with comorbid pain also showed a signi cantly shorter time to achieve treatment response and remission. The better antidepressant outcomes in depressed patients comorbid with pain indicated that ketamine works on the brain through mechanisms different from the mechanisms of common antidepressants. Interestingly, the pain group also showed mild pain during ketamine treatment, even after ketamine treatment. A systematic review reported that headache is the most common acute side effect after ketamine treatment, especially in patients given intravenous ketamine [36]. In the present study, pain symptoms were reported during 84 (17.9%) infusions from among the 468 total infusions of ketamine. Although most of them reported that the pain resolved shortly after dose administration, their VAS score, sensory index, affective index and PPI still re ected pain symptoms because these assessments covered a 24-h postinfusion period.
Ketamine showed analgesic effects in patients suffering from acute and chronic pain, as well as rapidly robust antidepressant effects in patients with MDD. Several clinical studies have supported that subanesthetic doses of ketamine may be ideal for the treatment of pain and depression comorbidities. Subanesthetic ketamine can reduce depressive symptoms in chronic pain patients, even in patients with refractory neuropathic pain syndromes [23,24]. Daily oral ketamine for 6 weeks also effectively improved depressive symptoms in patients with chronic pain with mild-to-moderate depression [26]. Furthermore, in animal studies, ketamine has been reported to relieve pain-induced depression, which is independent of its antinociceptive effect. The present study is the rst to examine the e cacy of repeated ketamine in depressed patients with pain. The results again proved ketamine's antidepressant and analgesic effects and gave rise to an interesting nding that depressed patients with pain achieved greater antidepressant outcomes than those without pain and took a shorter time to reach those outcomes.
Then, we further analyzed whether alterations in in ammatory cytokines were related to individual differences in ketamine's effects on comorbid depression and pain. We observed that most of the 19 in ammatory cytokine levels decreased from the mean change values after six infusions of ketamine treatment, consistent with our previous ndings in an overlapping sample [27]. By comparing changes in in ammatory cytokine levels after ketamine treatment, a greater decrease in IL-6 levels at 24 h after six infusions and greater decreases in IL-10, MIP-3α, IL-1β, IL-5 and IL-6 levels at two weeks after six infusions were observed in patients with pain than in patients without pain. We speculate that there is a relationship between downregulation of the in ammatory response and ketamine's superior antidepressant effects in patients with pain. Moreover, given that the depressed patients with pain exhibited higher plasma IL-1β, IL-6 and GM-CSF levels than the patients without pain before the ketamine intervention, it is likely that patients who have elevated in ammatory responses may more easily bene t from ketamine. In previous clinical studies, higher levels of IL-6 and IL-1β were reported as potential predictors of ketamine's antidepressant e cacy [37]. In an animal study, spared nerved ligation rats with a depression-like phenotype showed lower serum levels of IL-1β and IL-6 than nonresponders at baseline [34]. In addition, the results from rats subjected to inescapable electric shock suggested that peripheral IL-6 may contribute to resilience versus susceptibility to inescapable stress [38]. Thus, IL-6 was the only in ammatory cytokine that displayed a greater decrease immediately after ketamine treatment in the patients with pain than in the patients without pain.
Interestingly, correlations between changes in IL-6 levels and both antidepressant and analgesic effects were found in the depressed patients with pain at day 13; however, further analysis showed that ketamine's analgesic effect mediated the association between decreases in IL-6 levels and its antidepressant effect. Previous studies have also suggested that ketamine can decrease the expression of in ammatory cytokines in MDD patients, but the results regarding the relationship between changes in cytokine levels and antidepressant e cacy have been inconsistent. Chen et al. found that the decrease in levels of TNF-α after a single dose of ketamine in patients with MDD was correlated with antidepressant e cacy [39], while no association was found in Park's clinical study [40]. In combination with the present results, the downregulated in ammation, especially the decrease in IL-6 levels, may play a more direct role in ketamine's analgesic effect than its antidepressant effect. However, the precise mechanisms underlying the relationship between elevated in ammatory responses and susceptibility to the comorbidity of pain and depression are currently unknown. Further preclinical studies are warranted to determine the precise anti-in ammatory mechanism of ketamine in combined models of depression and pain.
This study was associated with several limitations. First, the patient sample was relatively small. The small sample size made it impossible to perform subgroup analyses by the area of pain. Second, seven participants lacked in ammatory cytokine data at 2 weeks after ketamine treatment. The third limitation was that in ammatory cytokines were measured only in peripheral blood, which does not directly re ect the in ammatory response in the brain.
Our study suggested that an elevated in ammatory response plays a critical role in the individual differences among depressed patients with or without pain. Ketamine showed great antidepressant and analgesic effects in depressed patients with pain, which may be related to its anti-in ammatory effect.
Further preclinical studies to address the precise anti-in ammatory mechanism of ketamine and future therapies based on such a mechanistic understanding can be developed to better serve those with a depression and pain comorbidity.   Change insensory index, affective index and present pain intensity in pain group and non-pain group.
Legend: * represents signi cant difference at given time point between pain group and non-pain group according to post hoc analysis (p<0.05). Abbreviations: PPI, present pain intensity.