Morphine-induced Immunosuppression Research Articles

Abstract PO-089: Opioid-mediated suppression of anti-tumor immunity via peripheral OPRM1 signaling in head and neck squamous cell carcinoma

Abstract Head and Neck squamous cell carcinoma (HNSCC) causes severe pain, beyond what is reported in other cancer types. Opioids are the current backbone of HNSCC pain treatment but may have an immunomodulatory effect in the presence of cancer; T cells express opioid receptors. We recently demonstrated that opioids may affect the efficacy of anti-PD1 monoclonal antibody (mAb) treatment in recurrent/metastastic HNSCC patients; opioid usage was associated with significantly lower progression free survival and overall survival as well as decreased CD8 T cells in the tumor microenvironment. We hypothesized that cancer immunosurveillance is impaired by the immunosuppressive actions of exogenous opioids via peripheral mu-opioid receptor (OPRM1) signaling. A syngeneic mouse oral cancer model was used to determine if analgesic morphine impacted the tumor-associated immune response during tumor progression and in response to anti-PD1 mAb; mouse oral cancer cell line 1 (MOC1) tumor-bearing mice were treated with vehicle or morphine (10mg/kg i.p., 2x daily for 4.5 days) and the tumor-associated immune infiltrate was assessed between groups using flow cytometry. Anti-PD1 mAb (250µg/mouse, 3 × 2 day interval) was initiated directly following morphine treatment. Co-administration of methylnaltrexone (MNTX), a peripheral OPRM1 antagonist used to confirm specificity and site of action of the morphine-induced effects. Finally, any direct effects of morphine on tumor cells were investigated in oral cancer cell lines using quantitative PCR and colorimetric proliferation assays. Morphine induced a 75±5% reduction in CD8+ T cells and a 90±3% reduction in CD19+ B cells in MOC1 compared to sham mice (n=6/group); morphine-induced immunosuppression was completely blocked with co-treatment of MNTX. Studies involving morphine administration prior to anti-PD1 treatment are currently underway but given the substantial suppression in infiltrating lymphocyte populations, efficacy is expected to be lessened. Lastly, Oprm1 expression was not detected at the mRNA level in mouse oral cancer cell lines. In vitro, 10µM morphine did not induce cell proliferation (proliferation relative to vehicle after 48hr; vehicle=100±8.3%, morphine=115.7±9.6%) or improve wound healing (wound closure after 24hr; vehicle=76.2±11.6%, morphine=78.5±11.4%). Taken together, our data suggest that morphine suppresses anti-tumor immunity via peripheral OPRM1 signaling in immune cells but not tumor cells. Further investigation is warranted to determine whether peripheral opioid blockade (i.e. MNTX) can slow tumor progression and improve response to cancer treatments while preserving centrally-mediated analgesia. Citation Format: Lisa A. McIlvried, Mona Yuan, Nicole L. Horan, Megan A. Atherton, Marci L. Nilsen, Dan P. Zandberg, Nicole N. Scheff. Opioid-mediated suppression of anti-tumor immunity via peripheral OPRM1 signaling in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-089.

Gypenosides ameliorate morphine-induced immunosuppression with an increased proportion of thymic T lymphocyte subsets and are involved in the regulation of the cAMP-CREM/CREB-IL-2 pathway

Gypenosides ameliorate morphine-induced immunosuppression with an increased proportion of thymic T lymphocyte subsets and are involved in the regulation of the cAMP-CREM/CREB-IL-2 pathway

Compound 511 ameliorates MRSA-induced lung injury by attenuating morphine-induced immunosuppression in mice via PI3K/AKT/mTOR pathway

BackgroundOpioids are widely used in clinical practice. However, their long-term administration causes respiratory depression, addiction, tolerance, and severe immunosuppression. Traditional Chinese medicine (TCM) can alleviate opioid-induced adverse effects. Compound 511 is particularly developed for treating opioid addiction, based on Jiumi Liangfang, an ancient Chinese drug treatment and rehabilitation monograph completed in 1833 A.D. It is an herbal formula containing eight plants, each of them contributing to the overall pharmacological effect of the product: Panax ginseng C. A. Meyer (8.8%), Astragalus membranaceus (Fisch.) (18.2%), Datura metel Linn. (10.95%), Corydalis yanhusuo W. T. Wang (14.6%), Acanthopanar gracilistμlus W. W. Smith (10.95%), Ophiopogon japonicus (Linn. f.) Ker-Gawl. (10.95%), Gynostemma pentaphyllum (Thunb.) Makino (10.95%), Polygala arvensis Willd. (14.6%). This formula effectively ameliorates opioid-induced immunosuppression. However, the underlying mechanism remains unclear. PurposeTo reveal the effects of Compound 511 on the immune response of morphine-induced immunosuppressive mice and their potential underlying molecular mechanism. This study provides information for a better clinical approach and scientific use of opioids. MethodsImmunosuppression was induced in mice by repeated morphine administration. Th1/Th2/Th17/Treg cell levels were measured using flow cytometry. Splenic transcription factors of Th1/Th2/Th17/Treg and outputs of the regulatory PI3K/AKT/mTOR signaling pathway were determined. Subsequently, methicillin-resistant Staphylococcus aureus (MRSA) was administered intranasally to morphine-induced immunosuppressive mice pretreated with Compound 511. Their lung inflammatory status was assessed using micro-computer tomography (CT), hematoxylin and eosin (H&E) staining, and enzyme-linked immunosorbent assay (ELISA). ResultsCompared to morphine, Compound 511 significantly decreased the immune organ indexes of mice, corrected the Th1/Th2 and Treg/Th17 imbalance in the immune organs and peripheral blood, reduced the mRNA levels of FOXP3 and GATA3, and increased those of STAT3 and T-bet in the spleen. It improved immune function and reduced MRSA-induced lung inflammation. ConclusionCompound 511 ameliorates opioid-induced immunosuppression by regulating the balance of Th1/Th2 and Th17/Treg via PI3K/AKT/mTOR signaling pathway. Thus, it effectively reduces susceptibility of morphine-induced immunosuppressive mice to MRSA infection.

Morphine-induced microglial immunosuppression via activation of insufficient mitophagy regulated by NLRX1

BackgroundChronic morphine exposure induces immunosuppression in the peripheral and central nervous system, resulting in susceptibility of patients to invading pathogens. Mitophagy is a crucial regulator of inflammation, and dysregulated mitophagy may cause immunosuppression, but whether mitophagy is linked with morphine-induced immunosuppression in the brain remains unknown. NLRX1 is the only mitochondrially localized NOD family receptor protein which serves as a critical regulator in immunity and mitophagy activation, but it remains an enigma how NLRX1 functions in the crosstalk between microglial inflammatory defense and mitophagy in the presence of morphine.MethodsPrimary microglia and astrocytes, BV2 and MA cell lines were utilized. Mice were stimulated with repeated morphine treatment to mimic chronic morphine exposure, and activation of mitophagy, lysosomal functions, and inflammation were assayed in specific brain regions and immune organs with or without NLRX1-silencing.ResultsMorphine induced microglial mitophagy in a LC3 (microtubule-associated proteins light chain 3)-dependent manner, which was mediated by NLRX1. Contrastingly, morphine impaired lysosomal functions, including generation, acidification and mitophagosome–lysosome fusion, thus leading to insufficient mitophagy activation in microglia. NLRX1-silencing inhibited mitophagy activity and rescued lysosomal functions including generation and acidification in microglia. The NLRX1-mediated incomplete mitophagy in microglial cells contributed to immunosuppression and vulnerability towards pathogenic challenge after morphine treatment. In vivo, NLRX1-mediated microglial mitophagy activation by morphine was mainly located in the murine brain cortex, striatum, and cerebellum, where NLRX1 functioned as a negative immune regulator and facilitated septic shock. Collectively, microglial immune responses to septic shock were amenable to NLRX1 silencing in the brain with morphine treatment.ConclusionMorphine activated insufficient mitophagy in microglia which was regulated by NLRX1, ultimately leading to host immunosuppression and susceptible conditions in the brain.

Effect of systemic injection of heterogenous and homogenous opioids on peripheral cellular immune response in rats with bone cancer pain: A comparative study.

Exogenous and endogenous opioids have been shown to modulate the immune system. Morphine-induced immunosuppression has been investigated extensively. However, the immune-regulating function of endogenous opioid peptides is unclear. The present study aimed to evaluate the difference in effects on cellular immune function between recombinant rat β-endorphin (β-EP; 50 µg/kg) and plant source morphine (10 mg/kg) via intraperitoneal injection treatment in a rat model of bone cancer pain. Walker 256 cells were injected into a tibial cavity injection to establish the bone cancer pain model. The paw withdrawal thresholds and body weights were measured prior to surgery, at 6 days after surgery, and following 1, 3,6 and 8 treatments. The spleen cells were harvested for detection of T cell proliferation, natural killer (NK) cell cytotoxicity, and the relative quantities of T cell subtypes (CD3+, CD4+ and CD8+ cells). Plasma levels of interleukin-2 (IL-2) were also determined. It was found that single or multiple treatments with β-EP (a homogenous opioid peptide) and morphine (a heterogenous opioid) had good analgesic effects on bone cancer pain, while the analgesia provided by morphine was stronger than that of β-EP. Treatment with β-EP 3, 6 and 8 times increased the body weight gain in the rat model of bone cancer pain, while morphine treatment had on effect on it. With regard to immunomodulatory functions, β-EP treatment increased T cell proliferation and NK cell cytotoxicity, and increased the relative quantities of T cell subtypes, but no effect on T cell secretion. However, morphine treatment decreased T cell proliferation and the levels of T cell subtypes. These data indicate that opioids from different sources have different effects on cellular immune function in vivo. A small dose of homogenous opioid peptide exhibited positive effects (analgesia and immune enhancement) on cancer pain. These results provide experimental evidence supporting the exploitation of human opioids for the treatment of cancer pain.

Involvement of the NLRP3 inflammasome in the modulation of an LPS-induced inflammatory response during morphine tolerance

BackgroundMorphine is widely used for its analgesic effects. In addition to its high potential for addiction and tolerance, morphine also induces immunosuppression. Inflammasomes, NLRP3 being the most characterized, is a platform for activation of pro-inflammatory cytokines, particularly IL-1β. We have explored the effects of lipopolysaccharide (LPS) during morphine tolerance on expression of the NLRP3 inflammasome and related inflammatory genes. MethodsMorphine-pellet administration was used to induce morphine tolerance in F344 rats. Control rats were given a placebo. On day 5, the animals received either saline or 250μg/kg LPS. LPS-induced protein expression of TNF-α, IL-1β, and IL- 6 was examined in the spleen of rats with and without morphine tolerance. A PCR array was used to examine LPS-induced expression of 84 inflammasome-related genes with and without morphine tolerance. ResultsLPS-induced IL-1β and TNF-α protein expression was significantly lower in the spleen of the morphine-tolerant animals than in the placebo-control animals. In response to LPS, expression of 27 genes, including NLRP3, TNF-α, IL-1β, and IL-6, was significantly increased, and expression of 3 genes was significantly decreased in both the morphine-tolerant and placebo-control groups compared to the saline-treated animals. However, there was only a 2.7-fold increase in NLRP3 expression in response to LPS in the morphine-tolerant rats compared to a 4.5-fold increase in the placebo-control animals. ConclusionOur data indicate that, in the morphine-tolerant state, LPS-induced expression of NLRP3 is suppressed and cytokine/chemokine expression is inhibited, which may be one of the mechanisms involved in morphine-induced immunosuppression.

MiR-155 modulates morphine-induced immunosuppression by targeting SK3 channel in microglia

MiR-155 modulates morphine-induced immunosuppression by targeting SK3 channel in microglia

A potential role for Citalopram as an immunoenhancer on morphine-induced immunosuppression (101.9)

Abstract Chronic pain patients are often challenged with depression stemming from the long-term physiopsychological effects of their condition. Consequently, chronic pain patients are often treated with morphine, which can induce immunosuppression, along with an antidepressant. The antidepressant, Citalopram, is a serotonin reuptake inhibitor that may have immunomodulatory effects. We investigated whether Citalopram has immunoenhancing effects on morphine-induced immune suppression. Adult mice were pretreated for 7 days with either saline or Citalopram (10 or 30 mg/kg, twice daily intraperitoneal injections), followed by immunosuppression by subcutaneous implantation of a 25 mg morphine pellet for 48 h. Spleen, thymus, and lymph nodes were harvested to analyze total cell numbers. Total splenocyte numbers in all Citalopram-treated animals were significantly higher than saline-treated animals, while no differences were found in the thymus or lymph nodes. More specifically, a high dose of Citalopram (30 mg/kg) resulted in significantly higher B cell numbers in the lymph nodes, where T cell and NK cell numbers were not different. Interestingly, splenic lymphocyte populations did not differ between treatments. In summary, Citalopram may have a specific potential in the attenuation of morphine’s immunosuppressive effect by enhancing splenocyte numbers and lymph node B cell populations.

Involvement of RelB, a member of the NF-κB family, in morphine-induced immunosuppression in mouse

Involvement of RelB, a member of the NF-κB family, in morphine-induced immunosuppression in mouse

Chimeric DNA Vaccine Reverses Morphine-Induced Immunosuppression and Tumorigenesis

Although long-term use of morphine has been shown to promote tumor growth, the question whether tumorigenesis occurs as a result of an immunosuppressive effect remains to be investigated. In mice rendered tolerant to morphine, the efficacy and mechanism of a vaccination to rescue morphine-induced immunosuppression and prevent tumor growth was assessed both in vitro and in vivo. Herein, we found that morphine-injected mice exhibited higher tumor growth rates and lower percentages of CD8+ T lymphocytes. The mechanism of morphine suppression of immunity might be through the suppression of E7-specific CD8+ T lymphocyte proliferation and the promotion of apoptosis of these cells by the Bcl-2 and Bax pathways. The suppressive effect of E7-specific CD8+ T lymphocytes by morphine could be reversed by naloxone. We have previously shown that calreticulin linked with E7 (CRT/E7) could enhance the CD8+ T cell response and the anti-tumor effects (W. F. Cheng et al. (2001) J. Clin. Invest. 108, 669–678). CRT/E7 DNA vaccine could overcome the immunosuppressive effect of morphine and suppress tumor growth. Our findings reveal that long-term morphine treatment dose-dependently promotes tumor growth and a DNA vaccine may serve as a useful approach to treat the profound immunosuppressive function and prevent tumorigenesis after long-term morphine treatment.

The immunosuppressive effects of chronic morphine treatment are partially dependent on corticosterone and mediated by the μ-opioid receptor

Wild-type and mu-opioid receptor knockout (MORKO) mice were used to investigate the role of corticosterone (CORT) and the mu-opioid receptor (MOR) in chronic morphine-mediated immunosuppression. We found that although plasma CORT concentrations in CORT infusion (10 mg/kg/day) and morphine-pellet implantation (75 mg) mice were similar (400-450 ng/ml), chronic morphine treatment resulted in a significantly higher (two- to threefold) inhibition of thymic, splenic, and lymph node cellularity; inhibition of thymic-lymphocyte proliferation; inhibition of IL-2 synthesis; and activation of macrophage nitric oxide (NO) production when compared with CORT infusion. In addition, results show that the inhibition of IFN-gamma synthesis and splenic- and lymph node-lymphocyte proliferation and activation of macrophage TNF-alpha and IL-1beta synthesis occurred only with chronic morphine treatment but not with CORT infusion. These morphine effects were abolished in MORKO mice. The role of the sympathetic nervous system on morphine-mediated effects was investigated by using the ganglionic blocker chlorisondamine. Our results show that chlorisondamine was able to only partially reverse morphine's inhibitory effects. The results clearly show that morphine-induced immunosuppression is mediated by the MOR and that although some functions are amplified in the presence of CORT or sympathetic activation, the inhibition of IFN-gamma synthesis and activation of macrophage-cytokine synthesis is CORT-independent and only partially dependent on sympathetic activation.

Effect of morphine and electro-acupuncture (EA) on apoptosis of thymocytes.

The influence of morphine and EA on the apoptosis of thymocytes were studied to investigate the posibility of its involvement in the mechanism of morphine-induced immunosuppression and the regulatory effect of EA on it. 1h after injecting 50 mg/kg morphine subcutaneously into 3-wk old Balb/c mice continually twice a day for 5 days, thymus was collected and the apoptotic cell was detected by a method of terminal deoxynucleotidyl transferase-meditaed dUTP nick end-labeling(TUNEL). The results showed that morphine significantly enhanced the percentage of TUNEL positive cells inside thymus with an appearing of apoptotic DNA ladder after 24 h incubation. Treating mice with EA of "Zusanli(St.36)" and "Lanwei(Ext.33)" for 1h after morphine administration decreased the percentage of TUNEL positive cells. EA also showed an regulatory effect on the increased the expression of CPP32 and decreased the expression of Bcl-2 by morphine. The significant enhancement of hypothalamic CRF and plasma ACTH level by morphine and the antagonize effect of EA on it suggested a possible role of Hypothalamus-pituitary-adrenal (HPA) axis played in the apoptosis of thymocytes by morphine and the regulatory effect of EA.

Immunomodulation mediated by microinjection of morphine into the periaqueductal gray matter of the mesencephalon.

Opioids such as morphine have profound suppressive effects on the immune system. Many studies have shown that the immunocompetence of macrophages, NK cell activity, and T and B lymphocyte functions are altered following administration of opioid agonists(1-6) There is growing evidence suggesting that there exists a complex, bidirectional link between central nervous system (CNS) and immune system(7). Elucidation of the mechanism(s) through which central opioid system induces immunosuppression is complicated by multiple communicative pathways among the nervous, endocrine and immune systems. It has been proved that the opioid receptors(3, 8-11) specifically e, but not δ or K receptors in the CNS(12, 13) play an important role in morphine-induced immunosuppression. The periaqueductal gray (PAG) matter of the mesencephalon has been reported to be one of the primary neural foci for the action of centrally administered morphine in eliciting the suppression of splenic NK cell activity(3). This immunosuppression has been hypothesized to be due to the activation of the hypothaiamic-pituitary-adrenal (HPA) axis or the sympathetic nervous system (SNS) or both(3, 14). Although it has long been known that the PAG is involved in regulation of many physiological functions including pain, fear and anxiety, autonomic regulation, vocalization and lordosis, aggressive behavior, reproductive behavior as well(15), the relationship between the PAG and the immunity is still unclear. This chapter will document the current knowledge of the effects of morphine microinjected into the PAG on neuroendocrine and immune systems.

Role of Adrenal Cortical Activation in the Immunosuppressive Effects of Chronic Morphine Treatment*

Implantation of a 75-mg morphine pellet in sham-adrenalectomized male C3H/HeN mice resulted in significant elevations of serum corticosterone levels within 6 h. Corticosterone levels remained elevated (3- to 4-fold) for 72 h and had returned to normal by 120 h postimplantation. Within 48 h of pellet implantation, morphine-pelleted mice exhibited marked reductions in spleen (35%) and thymus weight (56%) relative to values in placebo-pelleted controls. In addition, adrenal hypertrophy was observed in the morphine-pelleted shams (50% increase in adrenal weight relative to placebo. The magnitude of splenic and thymic atrophy was reduced by about 50% in adrenalectomized morphine-pelleted mice (17% and 22% reductions, respectively) compared to that in adrenalectomized mice implanted with placebo pellets. Lymphocyte proliferative responses to the T-cell mitogen Concanavalin-A and the B-cell mitogen bacterial lipopolysaccharide were also significantly reduced in the morphine-pelleted sham mice. Morphine-induced suppression of Concanavalin-A- or lipopolysaccharide-stimulated lymphocyte proliferation was absent in adrenalectomized mice. Effects similar to adrenalectomy (e.g. lessening of magnitude of morphine-induced suppression of lymphoid organ weight and lymphocyte proliferation) were found in morphine-pelleted mice given the glucocorticoid receptor antagonist RU-486 at a dose of 10 mg/kg, twice daily. These studies imply that morphine-induced immunosuppression is at least in part mediated by the increase in serum corticosterone levels after implantation of the morphine pellet.