Chronic Adrenergic Stress And Generation Of Myeloid-derived Suppressor Cells: Implications For Cancer Immunotherapy in Dogs

Abstract

Recent studies have highlighted a key role played by the sympathetic nervous system (SNS) and adrenergic stress in mediating immune suppression associated with chronic inflammation in cancer and other diseases. The connection between chronic SNS activation, adrenergic stress and immune suppression is linked in part to the ability of catecholamines to stimulate the bone marrow release and differentiation of myeloid-derived suppressor cells (MDSC). Rodent model studies have revealed an important role for β-adrenergic receptor signalling in the suppression of cancer immunity in mice subjected to chronic stresses, including thermal stress. 

Importantly, therapeutic blockade of beta-adrenergic responses by drugs such as propranolol can partially reverse the generation and differentiation of MDSC, and partly restore tumour immunity. Clinical trials in both humans and dogs with cancer have demonstrated that propranolol blockade can improve responses to radiation therapy, cancer vaccines and immune checkpoint inhibitors. Thus, the SNS stress response has become an important new target to relieve immune suppression in cancer and other chronic inflammatory conditions.

The sympathetic nervous system is an important part of the body that controls many autonomic functions such as heart rate, Pukager number and breathing. At the same time, the sympathetic nervous system is also closely related to immunity. Although physiologically two separate systems, they are highly interdependent.

When we encounter emergencies such as fear, stress, and danger, the sympathetic nervous system is aroused and releases adrenaline and norepinephrine. These neurotransmitters speed up the heartbeat and raise blood sugar levels in response to the body's needs. At this time, immune function will be suppressed to a certain extent to ensure that the energy and resources of the sympathetic nervous system are allocated preferentially.

Under normal circumstances, however, the sympathetic nervous system is not always active. When it enters a state of rest and recovery, it releases transforming growth factors and rheumatoid factors, which stimulate the immune system.

In addition, the sympathetic nervous system can also regulate the number, type and function of immune cells through the action of neurotransmitters and receptors. For example, noradrenaline can inhibit T cell immune response through the interaction between α2-adrenoceptorβ and actin, while β2-adrenoceptor can stimulate antibody response and natural killer cell activity, and enhance human immunity.

Overall, the sympathetic nervous system and immunity are closely related. A moderate state of sympathetic arousal can regulate immune function and provide some assistance for the body's self-protection. Therefore, we should try to avoid excessive stress and anxiety and maintain a positive mood to maintain a healthy immune state of the body. From this point of view, we need to improve our immunity. Cistanche can significantly improve our immunity because the polysaccharides in Cistanche can regulate the immune response of the human immune system, improve the stress ability of immune cells, and enhance immunity. Bactericidal effect on cells.

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KEYWORDS

cytokines, immune cells, macrophage, norepinephrine, thermal stress.

1 | ADRENERGIC STRESS AND IMMUNE SUPPRESSION IN CANCER

Chronic inflammatory states associated with cancer activate both the sympathetic nervous system (SNS) and the hypothalamus-pituitary-adrenal (HPA) axis. Previous studies linking chronic stress to immune suppression have focused primarily on the HPA and cortisol release since elevated cortisol concentrations are broadly suppressive.

However, there is now a growing awareness that SNS and adrenergic stress also play an important role in immune suppression associated with inflammation in cancer.1–7 The major mediators of SNS-associated immune suppression are catecholamines, primarily norepinephrine (NOR) and epinephrine, acting through beta-adrenergic receptors (β-AR).4,8–11 These receptors are widely expressed in normal cells, including many immune cells. Myeloid cells and their functions are particularly affected by NOR signalling through β-ARs, and induction of their immune suppressive states following sustained β-AR signalling thereby indirectly suppressing tumour-adaptive immune responses, particularly those of T cells. Therefore, this review will focus on the role of the SNS and adrenergic stress in mediating immune suppression in cancer and chronic inflammation, and on strategies to target this pathway pharmacologically.

Initiation of the SNS-adrenergic stress response begins when the amygdala is activated by exposure to stressors from chronic inflammation, especially pro-inflammatory cytokines such as TNF-α and IL-6.12–14 The amygdala then transmits signals to the locus coeruleus which in turn activates sympathetic neurons in the spinal cord, which ultimately innervate the adrenal medulla and in some cases tumour cells directly (Figure 1). Activation of this pathway triggers the release of catecholamines (principally norepinephrine (NOR) and epinephrine) from the adrenal medulla into the bloodstream. These circulating catecholamines, and those also released at nerve endings, bind to two adrenergic receptors, β1-AR and β2-AR2, which are the primary signalling molecules for the immune suppressive effects of adrenergic activation.11,13

2 | ADRENERGIC STRESS AND THE CANCER CONNECTION

Understanding the connection between SNS activation and adrenergic stress and cancer immunity resulted from serendipitous observations made in rodent cancer studies.15–20 Repasky and others observed that tumours in mice grew more slowly when mice were housed at their preferred higher ambient temperatures (thermoneutral; 30
C) rather than at the customary human comfortable room temperatures (e.g., 22
C), which are thermally stressful for mice.17,21– 25 Thus, typical mouse husbandry conditions subject mice to chronic hypothermal stress, which in turn activates the SNS and catecholamine release, triggering suppression of tumour immunity, leading to accelerated tumour growth and metastasis.21,25,26 These seminal observations thus provided the first experimental evidence linking SNS stress and suppressed tumour immunity.

Subsequent studies have provided further insights into the catecholamine-immune suppression connection. Recently, it was reported that signalling through beta-adrenergic receptors (specifically β2-AR) on myeloid cells leads to the mobilisation of MDSC from the bone marrow and into the bloodstream.4,8 Sustained exposure to elevated concentrations of catecholamines also promoted the further differentiation of MDSC into more immune suppressive polymorphonuclear (PMN)-MDSC and monocytic (M)-MDSC and macrophages within tumour tissues and secondary lymphoid organs, including the spleen.4,8,27 Genetic ablation of the β2-AR receptor partially restored tumour immunity in mice, thus confirming the key role of this receptor in immune regulation. Importantly, pharmacologic blockade of β1-AR and β2-AR by the non-selective beta-blocker propranolol also restored tumour immunity, an effect that has been recapitulated in multiple murine models.8,27–30

Studies also identified STAT3 as the key signalling pathway in myeloid cells activated by β-AR signalling in response to SNS stress.4,8,27,28 Activation of the STAT3 pathway triggers several downstream effector pathways, which lead to the acquisition of additional immune suppressive functions by MDSC and tumour-associated macrophages.31 These upregulated immune suppressive pathways include VEGF, IL-4 and IL-5, CCR4 and FoxP3 pathways.8 Treatment with propranolol blocks β-AR-mediated activation of STAT3 and thereby prevents MDSC and macrophages from acquiring a fully immune suppressive phenotype.31 It is also important to note that the effects of pharmacologic β-AR blockade are reversible, such that drug exposure must be sustained to generate and maintain the full effects of immune function restoration in animals with cancer.

3 | MYELOID-DERIVED SUPPRESSOR CELLS AND THEIR ROLE IN CANCER

Myeloid-derived suppressor cells consist primarily of myeloid cell populations that resemble immature neutrophils and monocytes, released from the bone marrow in response to chronic inflammation, including inflammation associated with cancer and chronic infections.32–36 Expanded populations of MDSC can be detected in the circulation and secondary lymphoid organs and tumour tissues in rodents, dogs and humans with diverse cancers (Figure 2).33–35,37–40 Numbers of MDSC are also chronically elevated in chronic infections and other sources of chronic inflammation.33–35,41,42 

The generation and release of MDSC from the bone marrow are triggered in part by certain myeloid growth factors, including GM-CSF, G-CSF, IL-3 and IL-6.33,35 Under physiological conditions, MDSC serves an important regulatory role to suppress excessive inflammation and promote tissue healing.36,43 When MDSCs are mobilized and recruited in response to tissue damage or infection, they play an important role in resolving chronic inflammation.33,35,36 In the case of unremitting inflammation associated with cancer, accumulation of increasing numbers of highly immune suppressive MDSC interferes with the function of antigen-presenting cells such as dendritic cells, and directly and indirectly impairs the function of T cells, B cells and NK cells.33–35

Myeloid-derived suppressor cells are difficult to fully define by flow cytometry, as no single marker can be used to unambiguously identify MDSC. In humans, two populations of MDSC are identified, a CD11b+CD14CD66b+ population of cells defined as PMN-MDSCs, and a second population of CD11b+CD14+HLA-DRloCD15 cells defined as M-MDSCs.35,37,42 Markers of MDSC generally accepted in mice include CD11b+Ly6G+Ly6Clo for PMN-MDSC and CD11b+Ly6GLy6C+ for MMDSC.34,37 Less is known about canine MDSC, but several reports indicate that immunosuppressive MDSC populations can be identified as CD11b +MHC- cells that can be further subclassified, with PMN-MDSC classified as CADO48A+CD14-, and M-MDSC as CADO48A-CD14+. 38–40

Functional immune suppression is a key feature used to define MDSC. The most widely employed functional assay utilises co-culture of purified MDSC with mitogen-activated T cells, where T-cell suppression (proliferation, cytokine release) is the primary readout.44 This assay measures both the direct cell-to-cell contact effects of MDSC, as well as the immune suppressive effects of their secreted cytokines. These assays are important to functionally define MDSC, but are technically difficult to perform consistently, and often require the use of sorted cells for more accurate measurements.

Myeloid-derived suppressor cells utilise multiple mechanisms to suppress immune responses. One mechanism includes the release of immunosuppressive cytokines, such as TGF-β and IL-10, which act to directly suppress T-cell function.34,35,39,45 These cytokines also promote the generation and survival of regulatory T cells (Tregs), which are also very immune suppressive.27,37,46,47 Furthermore, MDSCs often overexpress programmed cell death ligand 1 (PD-L1), which potently suppresses T cells and NK cells expressing the cognate receptor PD-1.34,35,37,48 In some species, including dogs, MDSCs overexpress the enzyme arginase 1 (ARG-1), which leads to the depletion of L-arginine locally in tissues, triggering suppressed T-cell function and death.35,39,48,49 Expression of reactive oxygen species (ROS) by MDSC can also lead to direct T-cell killing.34,35,48 Finally, MDSC also inhibit tumour immunity by converting adenosine triphosphate (ATP) into adenosine, mediated by the ectonucleotidase enzymes CD39 and CD73. Unlike adenosine triphosphate, which is an immunologically active molecule that stimulates anti-tumour immunity, adenosine is an immunosuppressive molecule that suppresses T-cell mediated anti-tumour immunity. Thus, MDSC harness multiple, nonredundant pathways for suppressing immune responses in cancer and chronic infections.

4 | CLINICAL APPLICATIONS OF Β-BLOCKADE TO CANCER IMMUNOTHERAPY

Given the compelling evidence from rodent studies that adrenergic stress and catecholamine release play a key role in suppressing tumour immunity, there are multiple opportunities for targeting this pathway therapeutically. Rodent studies have demonstrated that the non-selective beta-blocker propranolol, a widely used cardiac and cardiovascular drug with a good safety margin, can interrupt the SNS-adrenergic-MDSC pathway and relieve tumour immune suppression.4,8,9,50 For example, in one study propranolol treatment reversed the recruitment of MDSC into tumour and lymphoid tissues, slowed the growth of subcutaneous tumours and blocked tumour metastases.8 In addition, propranolol administration together with tumour vaccines enhanced vaccine immunity and the accumulation of T cells within tumour tissues.51–54 Similar immune-enhancing effects were observed when propranolol was co-administered with ICI (immune checkpoint inhibitor) immunotherapy.4,26–28,55 Importantly, studies have also demonstrated that pharmacologic activation of the SNS pathway, using beta-agonists such as isoproterenol, stimulated tumour growth associated with greater mobilisation of MDSC from bone marrow and accumulation in tumour tissues. These findings thus provide further mechanistic evidence linking the adrenergic stress response and cancer immune suppression.8

Several large retrospective studies have highlighted the potential benefits of receiving beta-blockers on cancer survivorship, though it should be noted that not all studies have not identified a protective effect.56–58 Moreover, animal model data and clinical trial results from the use of propranolol as adjuvant immunotherapy when combined with radiation therapy have been published recently, prompting new interest in propranolol as an immunotherapy agent.59–63 The common biomarker immune correlate in these studies has been a reduction in the numbers of circulating MDSC, as well as reduced immune suppressive functionality. The immune-enhancing activity of β-blockade has also been reported when used in combination with tumour vaccines and with ICI. For example, in numerous rodent studies, raising the ambient temperature to rodent thermoneutral temperature (e.g., 30
C) was shown to augment the effectiveness of ICI immunotherapy.16–18,20– 22,25,26,64 Thus, there is considerable evidence that β-adrenergic blockade can be an important tool in stimulating tumour immunity in a variety of cancers. However, it is also important to note that not all beta-blockers may be equally effective in cancer immunotherapy, as more selective antagonists (e.g., β2-AR antagonists) may not be as effective as mixed β1-AR and β2-AR antagonists such as propranolol.

New tools and approaches including targeted immunotherapies are being developed for dogs with cancer, and dogs are now widely recognised as an important spontaneous large animal model for investigating new approaches to treating cancer in humans.65–68 The value of dogs as an immune cancer model stems in part from their ‘educated’ immune system, sculpted by prior vaccines, infections and age, much like the human immune system. In addition, the tumour biological behaviour in dogs more closely resembles human cancer responses, especially concerning tumour metastatic processes.68 For example, the canine cancer model has been used for immunotherapy trials in bone cancer and brain cancer.69–74

Although most research investigating MDSCs continues to be conducted in humans and mice, recent work has highlighted the opportunities for further studies to be conducted in dogs. For example, MDSC concentrations are positively correlated with mammary cancer burden in dogs, suggesting a possible opportunity to use MDSC levels as a biomarker to identify patients with advanced levels of disease and metastasis.75 Circulating numbers of MDSC were also increased in dogs with melanoma.39 A report by Goulart et al comprehensively described MDSC populations in dogs,76 including the use of RNA sequencing of both PMN-MDSC and M-MDSC populations to further defined their immunological properties.40 In addition to stimulating cancer metastasis, MDSC have also been implicated in promoting tumour angiogenesis and tumour immunosuppression in canine mammary carcinoma, in part through STAT3 signalling pathways.75

Several studies have examined the use of propranolol as a repurposed agent for treating cancer in dogs. Most studies to date have focused on the anti-angiogenic properties of propranolol, for the treatment of aggressive cancer in dogs known as hemangiosarcoma.62,77 In vitro, propranolol exerts significant anti-angiogenic activity against both non-transformed endothelial cells and hemangiosarcoma cells.62 As to the use of propranolol as an immune modulatory drug, we reported recently that treatment of dogs with brain tumours with propranolol (plus losartan) and a tumour vaccine resulted in a 20% objective tumour response rate, without additional treatment, along with a 60% overall biological response rate.78 Thus, there is clinical evidence that propranolol immunotherapy can exert clinical benefits in dogs with aggressive cancers and can also function as an effective vaccine adjuvant in dogs.

5 | CONCLUSIONS

Our understanding of how chronic adrenergic stress shape tumour immunity has advanced greatly in the past several years. Key pathways connecting catecholamines and beta-adrenergic signalling to MDSC mobilisation and STAT3-mediated differentiation have been identified, which provides new opportunities for pharmacologic manipulation of the SNS pathway for immunotherapy of cancer. Preclinical and clinical data from rodent, canine and human studies suggest that treatment with a non-selective β-blocker (propranolol) can exert anti-tumour immune activity when used alone, or in combination with radiation therapy, ICI therapy and tumour vaccination. These findings suggest that adrenergic blockade may be an effective and readily implemented strategy for improving cancer treatment in general, using either repurposed drugs or drugs specifically designed to target key steps in the adrenergic signalling cascade.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

Not applicable.

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