Altered Membrane Expression and Function of CD11b Play a Role in the Immunosuppressive Effects of Morphine on Macrophages at the Nanomolar Level

Morphine, one of the most efficacious analgesics, is effective in severe pain, especially in patients with concomitant painful cancers. The clinical use of morphine may be accompanied by increased immunosuppression, susceptibility to infection and postoperative tumor metastatic recurrence, and the specific mechanisms and clinical strategies to alleviate this suppression remain to be investigated. Expression of CD11b is closely associated with the macrophage phagocytosis of xenobiotic particles, bacteria or tumor cells. Here, we find that morphine at 0.1–10 nM levels inhibited CD11b expression and function on macrophages via a μ-opioid receptor (MOR)-dependent mechanism, thereby reducing macrophage phagocytosis of tumor cells, a process that can be reversed by thymopentin (TP5), a commonly used immune-enhancing adjuvant in clinical practice. By knocking down or overexpressing MOR on macrophages and using naloxone, an antagonist of the MOR receptor, and LA1, a molecule that promotes macrophage CD11b activation, we suggest that morphine may regulate macrophage phagocytosis by inhibiting the surface expression and function of macrophage CD11b through the membrane expression and activation of MOR. The CD47/SIRPα axis, which is engaged in macrophage-tumor immune escape, was not significantly affected by morphine. Notably, TP5, when combined with morphine, reversed the inhibition of macrophage phagocytosis by morphine through mechanisms that promote membrane expression of CD11b and modulate its downstream signaling (e.g., NOS2, IFNG, IL1B and TNFA, as well as AGR1, PDGFB, IL6, STAT3, and MYC). Thus, altered membrane expression and function of CD11b may mediate the inhibition of macrophage phagocytosis by therapeutic doses of morphine, and the reversal of this process by TP5 may provide an effective palliative option for clinical immunosuppression by morphine.


Introduction
Opioids such as morphine are widely used worldwide for clinical postoperative analgesia or other causes of severe pain. Clinical studies have found that the use of opioids (e.g., morphine, fentanyl) causes, in addition to some common side effects such as tolerance, addiction, respiratory depression, nausea, and constipation, an immunosuppressive effect that increases the risk of infection [1,2]. It is known that opioid addicts are at increased risk of infection, and the intersection between HIV infection and intravenous drug abuse has been identified [3,4]. In vitro and in vivo experiments, as well as epidemiological and

Nanomolar (nM) Morphine Directly Inhibits Macrophage Phagocytosis of Cancer Cells via MOR but Not by Altering the CD47/SIRPα Axis
Opioid receptor agonists or antagonists affect tumor growth by modulating macrophage function [31,32]. To further verify at the cellular level whether morphine reduces macrophage phagocytosis of tumors at therapeutic doses, we induced THP-1 cells into phagocytic macrophages with phorbol-12-myristate-13-acetate (PMA), followed by A549 cells labeled with the fluorescent dye probe Protonex TM Red 600, and co-cultured with macrophages for 24 h. The results were obtained by fluorescence intensity recorded by fluorescence microscopy photographs, and morphine at 10 nM significantly attenuated phagocytosis of A549 by macrophages compared to the controls (13.1 ± 3.2% vs. 34.7 ± 4.6%, 10 nM morphine vs. Ctrl, respectively, p < 0.01, n = 3, unpaired t-test, Figure 1A,B). Additionally, the phagocytosis of A549 by macrophages was examined by flow cytometry in the concentration range of 0.001 nM~10 µM, and it was found that morphine concentration dependently inhibited the phagocytic effect of macrophages (EC 50 (concentration producing half the efficacy) = 2.13 ± 0.16 nM, Figure 1C), which is the concentration required for morphine to activate MOR [33], indicating that direct inhibition of tumor phagocytosis by macrophages at nM levels of morphine is a possible phenomenon during clinical administration.  It has been shown that CD47, which is highly expressed on cancer cells, and regulatory protein α (SIRPα), a macrophage expressing receptor, form checkpoints for innate immunity, inhibiting macrophage-mediated phagocytosis and leading to immune escape of tumor cells [34]. We conjectured first whether morphine inhibits macrophage phagocytosis by altering the CD47/SIRPα axis. Therefore, we labeled tumor cells A549 and macrophages with APC-CD47 and PE-SIRPα flow antibodies, respectively, and their expression levels on the membrane were detected by flow cytometry (Figure 1D-G). 10 nM Morphine did not alter the expression of CD47 in A549 cells and SIRPα protein in macrophages (77.9 ± 0.9% and 65.9 ± 9.9%, for 0 and 10 nM morphine in the surface expressions of CD47, respectively, p > 0.05, unpaired t-test, n = 3, Figure 1D,E; 89.4 ± 0.6% and 87.7 ± 0.8% for 0 and 10 nM morphine in the surface expressions of SIRPα, respectively, p > 0.05, n = 3, unpaired t-test, Figure 1F,G). Moreover, treatment with 0.01 nM~10 µM morphine for 48 h did not change the proliferation viability of macrophages and A549 cells compared with the control group (p > 0.05, n = 3, one-way ANOVA followed by Dunnett's multiple comparisons test, Figure 1H,I). These results suggest that direct inhibition of cancer cells phagocytosis by macrophages at nM levels is not attained through direct proliferation-inhibiting effects on cancers, nor through the modulation of the macrophage-tumor immune escape pathway, at least not primarily through the mechanisms described above.

Morphine Inhibits the Phagocytic Effect of Cancer Cells by Decreasing the Membrane Expression of CD11b on Macrophages
CD11b mediates multiple monocyte/macrophage responses in the immune inflammatory response, and it is tightly associated with phagocytosis of macrophages [35]. Therefore, we speculated that morphine may affect the phagocytosis of macrophages by affecting their CD11b functions. A significant decrease in CD11b mRNA expression levels was detected by qRT-PCR in macrophages induced with morphine in the concentration range of 0.1 nM~1 µM for 48 h (p < 0.001, n = 4, one-way ANOVA followed by Dunnett's multiple comparisons test, Figure 2A).

Leukadherin-1 (LA1) Promotes Macrophage CD11b Activation to Alleviate the Inhibitory Effect of Morphine on Macrophage Phagocytosis
LA1 is a small molecule agonist of CD11b that enhances the innate immune response, and in vivo targeting of CD11b activation repolarizes TAMs and inhibits tumor growth by enhancing the pro-inflammatory immune response to tumors [23,36]. To verify whether LA1 could affect the inhibition of morphine on macrophage phagocytosis by regulating CD11b expression or function, we first examined the expression of genes related to the downstream signaling of macrophage CD11b activation and found that morphine inhibited the expression of macrophage immune activation genes, such as NOS2, IFNG, and IL1B, and TNFA mRNA expression was decreased (p < 0.05, n = 3, one-way ANOVA followed by Dunnett's multiple comparisons test, Figure 3B). The expression of immunosuppressive genes such as AGR1, PDGFB, IL6, STAT3 and MYC was also elevated after morphine administration (p < 0.05, n = 3, one-way ANOVA followed by Dunnett's multiple comparisons test, Figure 3A). Interestingly, the combination of LA1 and morphine reversed and restored the downstream signaling of macrophage CD11b activation compared to the morphine alone group (p < 0.05, n = 3, one-way ANOVA followed by Dunnett's multiple comparisons test, Figure 3A,B). followed by Dunnett's multiple comparisons test; Figure 2C,D). Therefore, morphine inhibited the phagocytosis of A549 cells by macrophages, probably because it suppressed the expression of macrophage CD11b. Furthermore, the inhibition efficiency (EC50 = 2.13 ± 0.16 nM) of phagocytosis was comparable to the inhibitory potency of morphine on CD11b expression (EC50 = 1.44 ± 0.39 nM, Figure 2E).

Nanomolar Morphine Inhibits Phagocytosis of Cancer Cells by Decreasing the Surface Expression of MOR and CD11b on Macrophage Membranes
Morphine at sub-nM to nM levels alone modulates macrophage CD11b function and reduces phagocytosis, while MOR is also expressed in macrophages, suggesting that morphine-mediated immunomodulation may be mediated through direct interaction with MOR on immune cells [37]. First, 48 h action of morphine on induced macrophages with a concentration range of 0.1 nM ~ 1 μM morphine revealed a significant decrease in mRNA accompanying the CD11b gene and a significant decrease in the OPRM (MOR) gene (p < 0.001, n = 4, one-way ANOVA followed by Dunnett's multiple comparisons test, Figure  2B).
These results confirm that LA1 promotes macrophage CD11b activation and reverses morphine-mediated inhibition of macrophage phagocytosis.

Nanomolar Morphine Inhibits Phagocytosis of Cancer Cells by Decreasing the Surface Expression of MOR and CD11b on Macrophage Membranes
Morphine at sub-nM to nM levels alone modulates macrophage CD11b function and reduces phagocytosis, while MOR is also expressed in macrophages, suggesting that morphine-mediated immunomodulation may be mediated through direct interaction with MOR on immune cells [37]. First, 48 h action of morphine on induced macrophages with a concentration range of 0.1 nM~1 µM morphine revealed a significant decrease in mRNA accompanying the CD11b gene and a significant decrease in the OPRM (MOR) gene (p < 0.001, n = 4, one-way ANOVA followed by Dunnett's multiple comparisons test, Figure 2B).
Then, morphine at concentrations of 0.01 nM~10 µM was applied to PMA-induced macrophages labeled with Anti-MOR antibody and FITC-anti-mouse IgG1 antibody, and flow cytometric results showed that morphine also significantly reduced the expression level of MOR on macrophage membranes (EC 50 = 5.53 ± 3.70 nM, Figure 4A). We then synthesized small interfering RNA (siRNA for MOR) and detected the transient knockdown efficiency by transient transfection of induced macrophages by qRT-PCR. The knockdown efficiency of the MOR siRNA group was above 85% compared with the control siRNA (p < 0.001, n = 3, unpaired t-test, Figure 4B, left). Knockdown of the OPRM gene significantly reduced the expression level of CD11b (p < 0.001, n = 3, unpaired t-test, Figure 4B, right).
Next, the surface expression of CD11b on macrophages after transient knockdown of OPRM gene was examined and the results showed that knockdown of the OPRM gene could significantly inhibit CD11b expression on the macrophage membrane compared with the control group (25.5 ± 4.6% vs. 42.2 ± 0.8%, MOR siRNA vs. Ctrl siRNA, respectively, p < 0.01, n = 3, unpaired t-test, Figure 4C,D).
These results suggest that morphine may contribute to its inhibition of macrophage phagocytosis by reducing the co-distribution of MOR and CD11b across the macrophage membrane.
These results suggest that morphine may contribute to its inhibition of macrophage phagocytosis by reducing the co-distribution of MOR and CD11b across the macrophage membrane.

The MOR Inhibitor Naloxone Reverses the Inhibitory Effect of Morphine on Macrophage CD11b and Rescues the Phagocytic Function of Macrophages
To further verify the relationship between opioid receptor activation and morphine inhibition of macrophage CD11b membrane expression and thus the restoration of its phagocytosis, we have chosen the opioid receptor antagonist, naloxone (NLX), for validation [38]. After pretreatment of macrophages with 10 µM NLX for 30 min followed by morphine at a final concentration of 10 nM for 48 h, NLX significantly increased membrane CD11b expression (34.4 ± 3.5%) compared with morphine at 10 nM alone (19.8 ± 3.8%) (p < 0.05, n = 4, t-test, Figure 5A,B).
These results suggest that the opioid receptor inhibitor naloxone can reverse the morphine-induced decrease in macrophage CD11b expression and restore the phagocytic effect of macrophages. Thus, morphine could reduce macrophage CD11b surface expression by activating MOR, which is another explanation for its inhibitory effect on macrophage phagocytosis. It is also possible that the decrease in membrane expression of MOR (endocytosis) is due to the sustained activation of MOR [39]. At the same time, co-administration of NLX and morphine suppressed the expression of CD11b immunosuppression-related genes AGR1, PDGFB, IL6, STAT3, and MYC (p < 0.05, n = 3, one-way ANOVA followed by Dunnett's multiple comparisons test, Figure 5G), and significantly increased the expression levels of NOS2, IFNG, IL1B, and TNFA, which stimulate immune activation (p < 0.05, n = 3, one-way ANOVA followed by Dunnett's multiple comparisons test, Figure 5H).
These results suggest that the opioid receptor inhibitor naloxone can reverse the morphine-induced decrease in macrophage CD11b expression and restore the phagocytic effect of macrophages. Thus, morphine could reduce macrophage CD11b surface expression by activating MOR, which is another explanation for its inhibitory effect on macrophage phagocytosis. It is also possible that the decrease in membrane expression of MOR (endocytosis) is due to the sustained activation of MOR [39].

Thymopentin (TP5) Reverses the Inhibitory Effect of Morphine on Macrophage Phagocytosis by Promoting the Surface Expression and Function of CD11b
It is also not well understood how morphine-mediated immunosuppression can be restored by combining it with other clinically used drugs. TP5 has very strong immunomodulatory activity and can significantly improve the cellular and humoral immune functions of the body, thus making it a potential drug for the treatment of primary or secondary immunodeficiency, tumors, and severe infections [28,30]. At the same time, TP5, as an immune booster for tumor treatment, can restore the immune system of tumor patients, and in combination with chemotherapy, it can likewise prevent immunosuppression caused by chemotherapy drugs.
Similarly, qRT-PCR results showed that the combination of TP5 and morphine also increased the expression levels of two genes, CD11b and OPRM ( Figure 6I,J). Pre-administration of TP5 also decreased the expression of CD11b immunosuppression-related genes AGR1, PDGFB, IL6, STAT3, and MYC (p < 0.05, n = 3, one-way ANOVA followed by Dunnett's multiple comparisons test, Figure 6K) and significantly increased the expression levels of NOS2, IFNG, IL1B, and TNFA, genes that stimulate immune activation (p < 0.05, n = 3, one-way ANOVA followed by Dunnett's multiple comparisons test, Figure 6L).
Together, these results suggest that TP5 reversed the inhibitory effect of morphine on macrophage phagocytosis, mainly by promoting membrane expression of CD11b and regulation of downstream signaling, a process in which nAchRs are also partially involved.

Discussion
Here, we find that morphine inhibited the phagocytic effect of macrophages on tumor cells in a system of macrophage and tumor cell co-culture, and that this inhibition was somewhat morphine concentration dependent (at nanomolar level). This inhibition of phagocytosis was not achieved by inhibiting the proliferative activity of macrophages as well as cancer cells A549, or the innate immunity CD47/SIRPα axis. Our results suggest that the inhibition of phagocytosis by morphine is due to its suppression of macrophage CD11b expression and function, and that the reduced phagocytic effect is consistent with a potency to suppress CD11b expression. In addition, LA1 can promote CD11b activation downstream to stimulate the expression of immune activation-related genes and inhibit immune suppression-related genes, thus reversing the immunosuppressive effects of morphine. Further studies with knockdown, antagonist inhibitors, and overexpression of MOR on macrophages suggest that morphine may inhibit phagocytosis of macrophages by reducing macrophage CD11b expression through MOR's surface expression and activation. We found that in combination with morphine, TPF, which is an immune-enhancing adjuvant, reversed the inhibitory effect of morphine on macrophage phagocytosis and restored the macrophage phagocytic effect, mainly by promoting CD11b expression and activation of downstream signaling. Thus, CD11b and MOR jointly contribute to the immunosuppressive effects of morphine, with an association between them, and TP5 can regulate both, thereby reversing the immunosuppression of morphine.
We suggest a possible mechanism for morphine-induced immunosuppression and decreased function, namely that morphine inhibits CD11b expression and function on macrophages via MOR activation, thereby suppressing macrophage phagocytosis. However, we do not know whether morphine only affects changes in CD11b expression during monocyte-to-macrophage conversion or also directly affects CD11b as a membrane receptor-mediated downstream immune-related signaling, which our current findings cannot distinguish. Additionally, whether there is a strong association between decreased membrane expression of MOR and decreased membrane expression of CD11b, and whether there is a direct interaction between the two, remains to be further investigated. The decrease in macrophage phagocytosis induced by nM levels of morphine may be due to two mechanisms: decreased expression of MOR and CD11b in the membrane, and activation of MOR receptors; it remains to be confirmed whether these two mechanisms interact or are causal. It is also not very clear whether the decreased membrane expression of MOR is caused by the sustained activation of MOR, which leads to a decreased surface expression of CD11b, or whether other factors mediate this process. Considering that the effect of LA1 activation does not fully restore morphine-suppressed CD11b surface expression, we speculate that there may be other mechanisms mediating this effect, which also need further investigation. This mode of action might not depend primarily on G-protein downstream signaling from the MOR receptors and could serve to modulate the immunosuppressive effects of morphine without affecting its analgesic effects.
The relationship between prolonged opioid intake and tumor development in noncancer chronic pain medication is not well understood, and there is growing evidence that patients in these settings are at risk for cancer [15]. The common dose of clinical postoperative morphine administered subcutaneously is 5-10 mg/dose and 10-40 mg/day in adults (up to 60 mg or more depending on the severity of cancer pain), and the blood concentration of morphine in vivo is~50-100 nM [41]; when administered intravenously, the common dose in adults for analgesia is 1-10 mg. Studies have shown that an intravenous infusion of morphine 10 mg has an initial blood concentration of 100-200 ng/mL, which converts to approximately 350-700 nM; the lowest effective analgesic blood concentration of morphine in vivo is 10-50 ng/mL, which converts to approximately 35-175 nM; blood concentrations in the range 1.8-38 nM were detected 1 h after oral administration of 10 mg morphine, with a mean blood concentration of 5.8 nM [42,43], peak serum concentrations of morphine reached 70 to 80 ng/mL within 10 to 20 min after intramuscular administration of 10 mg [44]. These concentrations of clinical use of morphine are within the range of the present study, which validates our hypothesis of clinical immunosuppression caused by morphine, of which immunosuppression of macrophages is one of the causes. In clinical practice, TP5 might be considered in combination with morphine, which can effectively circumvent the risk of reduced immune function due to long-term morphine use in oncology patients.
Another point is that immune cells have been shown to secrete acetylcholine, dopamine, and gamma-aminobutyric acid (GABA), which mediate immune signaling and play important immunomodulatory roles [45][46][47][48]. In this study, pretreatment with TUB significantly attenuated the reversal of morphine-induced CD11b surface expression decrease by TP5's treatments (Figure 7), suggesting a process in which nAchRs are also partially involved.
Another interesting question is whether the current results have the potential to be applied clinically to retain the analgesic effect of morphine while reducing its effect on immunosuppression. We have confirmed that there are no reports of TP5 affecting the clinical application of morphine analgesia. Only one study in an animal model found that TP5 treatment resulted in a slight decrease in sensitivity to painful stimuli in rats. TP5 enabled tumor-bearing animals to recover diminished behavioral activity and increased resistance to stressful stimuli and pain [49], suggesting that TP5 can exhibit a slight analgesic effect. Furthermore, the advantage of TP5 over naloxone is that it may only improve the immunosuppressive effects of morphine without affecting the analgesic effects of morphine.
Finally, for the clinical use of TP5, clinical administration is mainly intramuscular or subcutaneous and can be 50 mg per day [50]. A study evaluated the clinical efficacy and tolerability of high-dose intravenous TP5 in 16 patients with melanoma [51]. Patients received 1 g of TP5 intravenously every two days (approximately 300-800 micromolar blood levels) and then underwent a 5-week follow-up for evaluation. In this study, highdose intravenous TP5 administered three times a week enhanced immune function in patients with cutaneous and subcutaneous metastases from melanoma, with no associated side effects. Therefore, TP5 may reach relatively high concentrations at local administration sites, but concentrations in whole blood throughout the day in the average patient may be lower than those used in our tests. Relatively high doses of TP5 may be used as an immune-enhancing adjuvant to enhance the immune function of patients. Thus, although TP5 is also relatively safe at high concentrations, clinical applications to alleviate the immunosuppressive effects of morphine requires a reasonable dose and frequency of treatment to be attempted in a clinical setting.

Plasmids and siRNAs
The plasmid of pcDNA3.1-hMOR-WT was kindly gifted from Dr. Rui Wang. The siRNAs used in this study were purchased from Shanghai GenePharma (Shanghai, China).

SiRNA Mediated Knockdown
THP-1-differentiated macrophages were transfected (Lipofectamine TM RNAiMAX, 13778075, Invitrogen, CA, USA) using 100 nM of siRNA against MOR (MOR siRNA) or non-silencing siRNA (Ctrl siRNA) [52]. The medium was changed after 6 h of transfection and the culture was continued for 48 h. The OPRM knockdown efficiency of each oligomer was confirmed by RT-qPCR assay.

In Vitro Phagocytosis Assays
In vitro phagocytosis was performed using THP-1 cells, and these cells differentiated into macrophages by incubation with PMA for 48 h. THP-1 cells (2 × 105 cells/well) were seeded in 24-well plates and treated with morphine for 48 h. A549 cells were labeled with fluorescent dye probe ProtonexTM Red 600 (21207, AAT Bioquest, Pleasanton, CA, USA) in the dark for 30 min. The fluorescence of Protonex™ Red dye increases sharply as the pH decreases from neutral to acidic according to the manufacturer's protocol [55]. Then, 2 × 105 pHrodo-red-labelled target A549 cells were added to the macrophages for 24 h at 37 • C. Co-cultured cells were collected and washed with 0.5% BSA-PBS. High-resolution images were taken on an inverted fluorescence microscope (DMI3000 B, Leica, Wetzlar, Germany) and processed in ImageJ. Phagocytic activity was analyzed by flow cytometry (BD LSRFortessa™, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by FlowJo 10.6 software.

Cell Viability Assay
Cell viability was evaluated using the MTT assay kit (C0009, Beyotime, Shanghai, China) according to the manufacturer's protocol [57]. A549 cells were seeded in 96-well plates at a density of 1 × 104 cells/well. THP-1 cells were seeded in 96-well plates at a density of 5 × 104 cells/well and differentiated in macrophages as described. Cells were incubated overnight at 37 • C. Next, cells were treated with morphine and incubated for 48 h. Then 10 µL of MTT was added to each well and incubated for 4 h. The absorbance was measured at 570 nm using a microplate reader (VarioskanTM LUX, Thermo, Waltham, MA, USA). Cell viability was estimated by comparing the relative absorbance values with those of the untreated samples.

Statistical Analysis
Data are expressed as mean ± SEM. All experiments were performed independently, at least 3 times. Statistical analyses were performed as described in each corresponding legend. Differences between two groups were assessed by unpaired two-sided Student's t-test, and differences between multiple groups were assessed by one-way ANOVA and Dunnett's post hoc test. p less than 0.05 was considered statistically significant. Concentration-response relationships of CD11b and MOR were obtained by measuring fluorescence intensity in response to different concentrations of morphine, and all results that were used to generate a concentration-response relationship were from the same group. The data were fit to the Hill1 equation: I/I max = 1/[1 + (EC 50 /morphine) n ], where I is the normalized fluorescence intensity at a given concentration of ligands, I max is the maximum normalized fluorescence intensity, EC 50 is the morphine concentration producing half of the maximum fluorescence intensity, and n is the Hill1 coefficient.