Associations between peripheral inflammation and clinical phenotypes of bipolar depression in a lower-middle income country

Abstract Objective There has been increased interest in repurposing anti-inflammatories for the treatment of bipolar depression. Evidence from high-income countries suggests that these agents may work best for specific depressive symptoms in a subset of patients with biochemical evidence of inflammation but data from lower-middle income countries (LMICs) is scarce. This secondary analysis explored the relationship between pretreatment inflammatory markers and specific depressive symptoms, clinical measures, and demographic variables in participants with bipolar depression in Pakistan. Methods The current study is a cross-sectional secondary analysis of a randomized controlled trial of two anti-inflammatory medications (minocycline and celecoxib) for bipolar depression (n = 266). A series of logistic and linear regression models were completed to assess the relationship between C-reactive protein (CRP) (CRP > or < 3 mg/L and log10CRP) and clinical and demographic features of interest and symptoms of depression. Baseline clinical trial data was used to extract clinical and demographic features and symptoms of depression were assessed using the 24-item Hamilton Depression Rating Scale. Results The prevalence of low-grade inflammation (CRP > 3 mg/L) in the sample was 70.9%. After adjusting for baseline body mass index, socioeconomic status, age, gender, symptoms related to anhedonia, fatigue, and motor retardation were most associated with low-grade inflammation. Conclusions Bipolar disorder (BD) patients from LMICs may experience higher rates of peripheral inflammation than have been reported in Western populations with BD. Future trials of repurposed anti-inflammatory agents that enrich for participants with these symptom profiles may inform the development of personalized treatment for bipolar depression in LMICs.


Introduction
Bipolar disorders, (BD) I and II, have a global lifetime prevalence of 0.6% and 0.4%, respectively. 1Effective treatment of depressive episodes in BD remains a challenge and bipolar depression contributes to substantial functional impairment. 2 In the World Mental Health survey initiative, severe and very severe role impairment was reported by 75% of BD patients who experienced depression within the 12-months prior. 1 A major factor contributing to this impairment is the lack of efficacy of current treatments for bipolar depression, which can be as low as 23.8% for first-line pharmacotherapies. 3Mounting evidence suggests that a reactive immune system may contribute to the pathophysiology of bipolar disorder and that antiinflammatory agents may be efficacious bipolar depression. 4,5More recently, the largest clinical trial to date (n = 266) of anti-inflammatories for bipolar depression found that both minocycline and celecoxib were not superior to placebo in the treatment of bipolar depression. 6These conflicting results suggest that anti-inflammatories may only be effective for a subset of patients with bipolar depression.
Given the mixed findings from randomized controlled trials (RCTs) of anti-inflammatory agents in mood disorders, anti-inflammatories may not be effective for the syndrome of depression but may target specific symptoms that are more commonly associated with a reactive immune system.The association between a reactive immune system and specific symptoms has been studied in major depressive disorder (MDD) but less so in BD.In MDD, neurovegetative symptoms (eg, low energy, altered appetite) have been associated with inflammation independent of cognitive and mood symptoms, while cognitive (ie, impaired thinking) and mood symptoms (ie, anhedonia, feelings of worthless and guilt) were not associated with inflammation. 7Other studies have found that inflammation is associated with fatigue, sleep problems, depressed mood, and anhedonia in depression. 7,8 recent meta-analysis of 56 351 persons with MDD found that five symptoms, that is, little interest in doing things, sleep disturbance, changes in appetite, feeling like everything was an effort, and loss of energy, were associated with higher concentrations of plasma C-reactive protein (CRP). 9In another sample, specific neurovegetative symptoms of eating, appetite, and tiredness were associated with elevated CRP and not overall depression scores or severity. 10Symptoms of BD that have been associated with inflammation include disturbances in sleep, suicidality, cognitive dysfunction, and anhedonia. 11,12We have previously reported that in a sample from a lower to middle income country (LMIC) lower CRP was associated with higher rates of suicidality in BD; contrary to what has been found in Western populations. 13his suggests that the associations between inflammation and depressive symptoms in BD may differ in a LMIC setting.
LMICs represent nearly half of the world's population and over 80% of the disease burden attributed to mental disorders, yet are largely excluded from mental health research.Similarly, studies investigating the relationship between inflammation and depressive symptoms in LMIC populations are scarce.The objective of the current analysis is to: (a) Determine the prevalence of peripheral inflammation in a sample of adults with bipolar depression in a LMIC setting (Pakistan) and (b) To explore associations between plasma CRP, and clinical phenotypes of bipolar depression (demographic variables, clinical variables, and specific symptom scales) in this sample.

Study design
The original MINDCARE trial was a randomized, doubleblinded, multicentre 2 × 2 factorial design trial conducted in outpatient psychiatric clinics in Hyderabad, Karachi, Lahore, and Rawalpindi, Pakistan between May 1, 2016, and March 31, 2019.The study investigated the antidepressant effect of minocycline and celecoxib in adults with bipolar depression with a four-arm (2 × 2) factorial design.The study was approved by the institutional review board of Karachi Medical & Dental College (KMDC).Detailed methodology of the trial has been previously reported. 6articipants were between the ages of 18 and 65 with a DSM-5 diagnosis of BDI or BDII and concurrent major depressive episode.They were on stable medication for 4 weeks or greater prior to baseline assessment and provided informed consent.Females with child-bearing potential consented to using contraception and monthly pregnancy tests due to the teratogenic risk of minocycline and celecoxib.
Exclusion criteria included: serious physical health conditions including chronic infectious diseases; history of allergies to antiinflammatory drugs; currently using penicillin, anticoagulants, antibiotics, or other anti-inflammatory drugs; history of seizures; DSM-5 diagnosis of substance misuse within 3 months prior to screening; DSM-5 diagnosis of primary psychotic disorder; high suicide risk; experiencing manic or hypomanic symptoms (≥3). 6

Measurement of CRP
Blood samples were collected, and CRP was quantified using the Spinreact CRP Latex Agglutination test at baseline and week 12.

Definition of inflammatory status
We chose CRP >3 mg/L as a definition of low-grade inflammation based upon United States Centers for Diseases Control and Prevention and American Heart Association Guidelines, which considered CRP levels over 3 mg/L to be indicative of an active inflammatory response. 18,19This definition has also been utilized in other studies in BD. 20

Statistical analysis
All statistical analysis was conducted with SPSS-28 software.Descriptive statistics were used to explore the distribution of baseline demographic variables, clinical variables, and symptoms.All variables were analyzed grouped by inflammatory status (CRP < 3 mg/L or > 3 mg/L).Kruskal-Wallis test was used to determine any differences in these variables.For categorical variables, we compared difference in inflammatory status using Pearson chi-square tests.
We then completed a series of logistic regression analysis with inflammatory status (CRP < 3 mg/L or > 3 mg/L) as the dependent variable.Each model included one demographic, clinical, or symptom variable of interest as a covariate to assess its association with inflammatory status.SEC, gender, BMI, and age were selected a priori as covariates in each adjusted models given their known association with depressive symptoms and inflammation.
To assess CRP as a continuous variable, we also completed a series of similar linear regression models with log10CRP as the dependent variable.Each model included one demographic or symptom measure as a covariate to assess the association with baseline inflammation.The same covariates as the logistic regression models were included.

Baseline demographic and clinical measures by inflammatory status
A total of 266 patients were enrolled in the original study, which included 189 (71%) males and 77 (29%) females and mean age was 36.5 (10.4).Table 2 summarizes the baseline demographic, clinical, and symptom variables.Baseline CRP was available from 237 participants with a mean (SD) value of 5.94 (8.54) mg/L.A total of 168 participants (70.9%) had a baseline CRP > 3 mg/L and 69 (29.1%) participants had a baseline CRP < 3 mg/L.

Demographic variables
Age and SES were not equally distributed between the low-grade inflammation (CRP > 3 mg/L) group and noninflamed group (CRP < 3 mg/L).The mean age was higher in the low-grade inflammation group (mean: 37.9, SD:10.2) compared to the noninflamed group (mean:31.9,SD:9.5).There was a higher proportion of persons with a low SES in the noninflamed inflammation group (CRP < 3 mg/L) compared to the low-grade inflammation group (CRP > 3 mg/L) (Table 2).

Clinical variables
Clinical variables that were not equally distributed between the two groups were heart rate, blood pressure (systolic and diastolic), and proportion of patients taking an antipsychotic, benzodiazepine, or an anti-cholinergic.The mean heart rate for the low-grade inflammation group (CRP > 3 mg/L) was lower (mean: 82.8, SD: 9.1) compared to the noninflamed group (mean: 87.6, SD: 8.6).The mean blood pressure for the low-grade inflammation group was overall higher (systolic mean: 120.6, SD: 10.7 and diastolic mean: 83.4,SD: 9.4) compared to the noninflamed group (CRP < 3 mg/L) (mean:113.0,SD: 13.7 and mean: 78.9, SD: 11.8 respectively).There was a lower proportion of participants taking an antipsychotic or anti-cholinergic medication in the low-grade inflammation group (Table 2).There was a higher proportion of patients taking a benzodiazepine medication in the low-grade inflammation group (Table 2).
Symptom scale total scores CGI-S and the EQ-5D scores were not equally distributed between the two groups.The CGI-S (mean: 4.70, SD: 0.9) was lower in the low-grade inflammation group while the EQ-5D was higher in the low-grade inflammation group (mean: 45.96, SD: 13.7) compared to the noninflamed group (CRP < 3 mg/L).A higher CGI-S indicates higher severity and higher EQ-5D indicates a higher perceived health-related quality of life (Table 2).

Symptom clusters of symptom scales
Clusters of symptoms that were significantly higher in the lowgrade inflammation group include the anhedonia cluster, increased appetite cluster, somatic symptoms cluster, and energy cluster (Table 2).

Associations between low-grade inflammation and clinical variables logistic regression
The results of the logistic regression model controlling for BMI, SES, age, and gender are presented in Table 3 and Figure 1 and are summarized below.

Symptom clusters
There were four symptoms clusters that were associated with a higher likelihood of low-grade inflammation: anhedonia symptom clusters, somatic symptom clusters, energy symptom clusters, and pro-inflammatory symptom clusters (Table 1).The antiinflammatory cluster was negatively associated low-grade inflammation.The pro-inflammatory and anti-inflammatory symptom clusters are the individual items that were respectively associated with low-grade inflammation.

Log10 CRP linear regression models
The results of the linear regression with log10CRP as the dependent variable are presented in Supplementary Table 1 and Supplementary Figure 1 and summarized below.There were no demographic variables that were associated with the log10CRP in the linear regression models.A higher heart rate was associated with a lower log10CRP (B: À0.012, CI: À0.018 to À0.006, P < .001).Taking an antidepressant was associated with a higher log10CRP (B: 0.187, CI: 0.019 to 0.355, P: .03).Taking an anti-cholinergic medication was associated with a lower log10CRP (B: À0.163, CI: À0.321 to À0.006, P: .042).
A higher score in the CGI-S was associated with a lower log10CRP (B: À0.097, CI: À0.154 to À0.040, P = .001).The EQ-5D and HDRS total score were not associated with the continuous variable log10CRP.

Discussion
The current secondary analysis of the MINDCARE trial found a high prevalence (70.9%) of low-grade inflammation in a sample of Pakistani adults with bipolar depression.When controlling for SES, age, BMI, and gender, we found that elevated blood pressure, taking an antipsychotic or benzodiazepine, HDRS total, and EQ-5D scores were associated with low-grade inflammation.Symptoms that were associated with low-grade inflammation were related to anhedonia, gastrointestinal somatic complaints, carbohydrate craving, hypersomnia, decreased energy, and motor retardation.
The prevalence of low-grade inflammation in our sample is inconsistent with evidence from other settings.A recent study in a Han-Chinese BD sample (n = 430), found that rates of low-grade inflammation were 10.1%. 20This sample was similar in that they were treatment-seeking participants; however, less than half of the sample was currently depressed with only a moderate HDRS-17 (18.4) score.Other samples of BD, mostly from Western countries, have reported lower rates of low-grade inflammation ranging from 20% to 40% depending on phase of illness. 21,22An explanation for the higher rates of low-grade inflammation in our sample could be related to current medications.Previous studies have included a significant proportion of patients that were treatment naïve while all participants in the MINDCARE trial were taking at least two psychotropic medications at baseline.Previous research has shown that psychotropic medications have an influence on inflammatory markers. 21In the current sample antipsychotic medication was associated with lower rates of inflammation.This association has been previously reported in patients with schizophrenia, with antipsychotics leading to reduced expression of immune genes and down-regulation of cytokine levels.Given the cytokine role in anorexigenic responses, reduction in inflammation could contribute to change in eating behaviors and changes in metabolic parameters associated with antipsychotics. 23nother explanation may be related to relative SES.A recent meta-analysis in nonpatient populations in North America, found that higher rates of inflammation are found in persons with lower SES. 24Our sample, from a LMIC, represents a population with a relative lower SES compared to those in high-income settings, which may explain the high prevalence of low-grade inflammation.Though in our population, high SES was associated with low-grade inflammation, our sample was heavily biased toward low to medium SES.Within this spectrum of SES there may be complex factors that are unaccounted for and contributing to this association.For example, mounting evidence has shown that relative inequality, perceived SES, and neighborhood SES have a significant impact on overall health. 25,26Future work should explore the relationship between SES and inflammation within LMIC countries and compare this to findings from high-income countries.
Our study found several specific symptoms that are associated with both low-grade inflammation.Anhedonia has been repeatedly associated with a pro-inflammatory state in both BD and MDD as evidenced by cross-sectional studies and responsiveness of anhedonia to anti-inflammatory drugs. 12,27In our sample, we found that higher rates of anhedonia were seen in those with low-grade inflammation.In addition, low-grade inflammation was also associated with lower energy, hypersomnia, somatic symptoms, and psychomotor retardation.In the Han-Chinese population, low-grade inflammation was not associated with hypersomnia but was associated with leaden paralysis. 20The same study reported that low-grade inflammation was associated with a recent suicide attempt, while in our sample low-grade inflammation was associated with less suicidality as previously reported. 13As discussed above, one explanation may be the lower rates of depression in the Han Chinese sample vs our sample.Furthermore, both samples represent relatively unique groups with distinct psychosocial circumstances, lifestyle factors, and cultural practices.Sleep disturbances are a key feature of BD and have been linked to a pro-inflammatory state, with a bidirectional relationship. 12In the context of MDD, a large metaanalysis has shown increased CRP to be associated with little interest in doing things, sleep disturbance, changes in appetite, feeling like everything was an effort, and loss of energy. 9In a sample of MDD and BD, the effect of CRP on connectivity mediated significant relationships between CRP and anhedonia and motor slowing. 28Overall, our results are consistent with these studies, which reported that anhedonia, somatic, sleep, and fatigue-related symptoms are associated with higher CRP.

Limitations
This present study is an exploratory study with limitations.First, the study cannot conclude a causal relationship between inflammation and depressive symptoms in BD.We limited the current study to a cross-sectional analysis given the primary study did not report any anti-inflammatory effects or antidepressant treatment response of the study drugs over placebo.The current study only used CRP as the sole marker for inflammation.Other direct markers of inflammation, such as cytokines, may have a different correlation with the symptoms and provide insight about which subset of the BD population is responsive to anti-inflammatory agents.Most of the sample recruited to the MINDCARE trial were males hence the findings may not be generalizable to females.This is likely due to the under-representation of females in the treatment-seeking population in Pakistan.Studies suggest that there may be a gender difference regarding the association between CRP and symptom severity. 29Additionally, due to factors, including socioeconomic and lifestyle differences, the findings from Pakistan, a LMIC, are not necessarily generalizable to high-income countries or other LMICs.Future studies should explore psychosocial, lifestyle, and cultural factors that contribute to this relationship.

Conclusions
Our findings suggest a high prevalence of low-grade inflammation in Pakistani patients with bipolar depression.In this sample, there are several symptoms that may be more associated with inflammation such as anhedonia, hypersomnia, fatigability, and motor retardation.Further research validating these findings in larger samples is needed.If replicated, clinical trials of anti-inflammatory agents stratifying patients based upon this "inflammatory" clinical phenotype may improve treatment outcomes in a subset of patients with bipolar depression in LMICs.
Supplementary material.The supplementary material for this article can be found at https://doi.org/10.1017/S1092852923002316.