The Role of M1- and M2-Type Macrophages in Neurological and Infectious Diseases

Abstract

Macrophages have a critical role in the outcome of neurological diseases, including neurodegenerative, autoimmune, vascular and microbial diseases. Macrophage role ranges from beneficial to pathogenic depending upon genetics, other components of innate and adaptive immunity, lifestyle and macrophage targets: aggregated molecules or bacterial and viral pathogens. Macrophages are attracted by chemokines to migrate into the brain and remove or inactivate pathogenic molecules. In the patients with neurodegenerative diseases, macrophages target aggregated molecules, amyloid-β1–42 (Aβ) and P-tau in Alzheimer’s disease (AD), and superoxide dismutase-1 (SOD-1) in amyotrophic lateral sclerosis (ALS), but also have autoimmune targets. In AD and ALS patients, macrophages in the pro-resolution M1M2 state are adapted to brain clearance and homeostasis, whereas in the proinflammatory M1 state are modulate to an anti-viral and antibacterial role, which may be associated with collateral damage to tissues. In HIV-1 and CoV2 viral infections, macrophages in M1 state are anti-viral but also pathogenic through inflammatory damage to the heart and the brain. In neurodegenerative diseases, the natural substances polyunsaturated fatty acids (PUFA), vitamins B and D, energy molecules, and flavonoids have beneficial effects on macrophage transcriptome and functions for brain clearance, but the effects are complex and depend on many variables.

1. Introduction

Macrophages and dendritic cells are key players in activation of the innate and adaptive immune systems. In viral infections, such as HIV-1 and COVID-19, macrophages assume either a more protective or a pathogenic role depending on their classification.

Macrophages are polarized by cytokine signaling from CD4 T cells into the M1 or the M2 type. M1 macrophages are classically activated by interferon-γ (INF-γ), whereas M2 macrophages are activated by interleukin-4 (IL-4) and IL-13 [1]. M1 macrophages have a role in combating infection, whereas M2 macrophages in supporting homeostasis. This inflammatory response, while beneficial against microbes and aggregated molecules, when hyper-activated can lead to tissue damage in the lungs, heart, and brain.

Under conditions of high microbial activity, M1 promote inflammation, synthesize nitric oxide, and induce cytokine production IL-6, IL-12 and tumor necrosis factor (TNF). Type M2 macrophages secrete arginase-I, IL-10 and TGF-β and other anti-inflammatory cytokines promoting wound healing but also contributing to tumor growth. M1 macrophages phagocytize microbes and initiate an adaptive immune response by T cells while M2 macrophages induce collagen repair to maintain tissue repair [1].

The roles of M1 macrophages in opposing the disease and the M2 macrophages in promoting homeostasis, while evident in infectious diseases, is less evident in neurodegenerative diseases. Macrophages of AD patients were classified according to the ratio of cluster of differentiation (CD) surface markers: CD54+ CD80/CD163+ CD206 [2] as follows: the inflammatory M1 macrophage type has the ratio > 4, the anti-inflammatory M2 type has a ratio < 1. Both M1 and M2 are associated with progression of dementia. In AD patients, however, the intermediate M1M2 phenotype with a ratio between 1 and 4 is a pro-resolution type promoting homeostasis and protection against dementia. Infection or other stress can cause an imbalance in the expression of these two macrophage types, as seen by others [3].

Macrophages were first appreciated in tuberculosis, where the dual role of macrophages in containing vs. replicating Mycobacterium tuberculosis (Mtb) has been known for a century. A successful response to mycobacterial infection is a granuloma consisting of lymphocytes, macrophages, Langhans giant cells (fused macrophages around mycobacteria) and fibroblasts [4].

In recent years, the role of macrophages in neurodegenerative diseases, Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS), has been appreciated as a result of immunochemical demonstration of central nervous system (CNS) invasion by peripheral blood monocyte/macrophages (MM) in AD patients [5]. Macrophage targets in AD are amyloid-beta (Aβ) and P-tau, while in ALS macrophage targets are aggregated superoxide dismutase (SOD-1) and microbial nucleic acids. Autologous DNA released into the cytoplasm could be a target in the cGAS-STING pathway [6].

This chapter will discuss the integral role and modulation of macrophage role in the therapy of neurodegenerative and infectious diseases. The natural substances polyunsaturated fatty acids (PUFA), circuminoids, vitamin D, and polyphenols improve macrophage transcriptome and functions for brain clearance in neurological diseases, but their mechanisms depend on many variables that are under study. The effects of natural substances on macrophage transcriptome and clearance of Aβ and P-tau could potentiate therapeutic outcome of monocl0nal antibodies, which increase phagocytosis but not degradation or export of Aβ and P-tau.

2. Macrophages of Alzheimer’s disease patients fail in clearance of the AD brain

In AD patients, microglia are the first responders to accumulation of Aβ in the brain, which attract monocyte/macrophages (MM) from the blood into the CNS by chemokines, in particular CCL5 (RANTES) [7]. In non-demented individuals, post-mortem immunochemistry and results from a blood-brain barrier (BBB) model suggest that MM migrate across BBB in both directions, clear Aβ in plaques, degrade or export Aβ from the brain without disruption of BBB or accumulation of macrophages around vessels [5, 8], and deposit Aβ into cervical nodes or further downstream [9]. MM of healthy subjects with the pro-resolution M1M2 phenotype effectively upload Aβ into endosomes, fuse endosomes with lysosomes, and degrade Aβ by lysosomal enzymes. MM of AD patients are defective in internalization and degradation of Aβ in endosomes [8].

In the AD brain, MM immigrate into CNS through disrupted tight junctions between endothelial cells. In addition, opening of tight junctions in the blood-brain barrier (BBB) permits brain entry of fibrin and components of the complement. In the brain parenchyma, AD macrophages invade the plaques but upload Aβ only on its cell surface, do not transport Aβ into endosomes and fail to degrade Aβ. Clearance of individual Aβ plaques by macrophages is functionally stochastic, thus some plaques are not fully cleared [5]. After uploading Aβ, MM migrate to vessels but fail to emigrate across BBB, suffer apoptosis and release Aβ into vessels, which develop cerebro-vascular angiopathy (CAA) (Figure 1) [10].

Figure 1.

Macrophage pathway in the AD brain. Created with BioRender.com.

2.1 Natural substances have mixed results in prevention of Alzheimer’s disease

Natural substances, prominently PUFA, have a long history in prevention of AD in the patients with mild cognitive impairment (MCI). PUFA supplementation had positive cognitive effects in MCI patients, but only in those with very mild MCI [11]. A large controlled clinical trial of PUFA supplementation, however, did not slow the rate of cognitive and functional decline in patients with mild to moderate Alzheimer disease [12].

Nevertheless, proven effects of PUFA on macrophage transcriptome and functions designate PUFA for immunotherapeutic studies controlled for baseline immune phenotype, APO E genotype, stage of disease, diet and lifestyle using a high-quality omega-fatty acid preparation protected against oxidation [2]. In an uncontrolled study of mild cognitive impairment (MCI) patients supplemented by the omega-3 fatty acid drink Smartfish, macrophage phenotype changed from either a highly inflammatory M1 or a very low non-inflammatory M2 to the desired pro-resolution M1/M2 (Table 1) [2]. Importantly, in this study, PUFA supplementation improved the cognitive status of MCI/AD patients for 4–17 months and protected against dementia for 4.5 years in certain patients [13]. In vivo supplementation and in vitro stimulation by the Smartfish drink stimulated the pro-resolution M1/M2 macrophage type with increased phagocytosis and energy [14].

	AD patients	PUFA-supplemented AD patients
M1-type macrophages	↑	↓
M2-type macrophages	Variable	↑
M1/M2 pro-resolution-type macrophages	↓	↑↑
MGAT3 transcription	↓	↑↑
Inflammation	↑↑	↓
Aβ phagocytosis by macrophages	↓	↑

Table 1.

The effects of PUFA-supplementation on Alzheimer’s disease (AD) patients.

A clinical study of supplementation with a high dose of vitamin B complex, including vitamins B6 (pyridoxine), B9 (folic acid), and B12 (cobalamin), showed a reduction in the rate of brain atrophy in MCI patients with elevated total homocysteine [15].

2.2 Alzheimer’s disease patients’ macrophages have transcriptomic defects that underlie their defective function

AD brain cells and macrophages lack energy due to oxidative damage and nitrosylation by reactive oxygen species (ROS) of the glycolytic enzyme transcripts GAPDH, PKM1, and PDH, and the citric acid cycle enzymes ACO2 and OGDH [16]. The defects in macrophage transcriptome in protein-coding and regulatory sequences for energy, glycosylation, unfolded protein response, glymphatic system, Toll-like receptors, and other genes lead to macrophage malfunction and apoptosis [14]. Macrophages exhibit a faulty unfolded protein response (UPR) to endoplasmic reticulum (ER) stress from Aβ phagocytosis. These defects lead to macrophage apoptosis and inflammation [14].

2.3 Natural substances have positive effects on macrophage biochemistry

The anti-inflammatory molecule bisdemethoxycurcumin up regulated MGAT3 transcripts and increased macrophage phagocytosis of Aβ. 1,25 dihydroxy vitamin D3 potentiated the 4,4-diisothiocyanostilbene-2,2-disulfonic acid-sensitive chloride channel ClC-3 currents essential for Aβ phagocytosis [17]. A clinical study of supplementation with a high dose of vitamin B complex, including vitamins B6 (pyridoxine), B9 (folic acid), and B12 (cobalamin), showed a reduction in the rate of brain atrophy in MCI patients with elevated total homocysteine [15].

RNA-seq analysis of single blood cell types in PUFA-supplemented MCI patients showed up-regulation of the transcripts for glycolysis, tricarboxylic acid cycle, OX-PHOS, nicotinamide dinucleotide (NAD⁺) synthesis in monocytes, neutrophils, regulatory T cells, memory CD4 and CD8 T cells, and NK cells, but the most consistent effects across the whole spectrum were in monocytes/macrophages (MM). Importantly, the supplementation showed that PUFA provide energy for immune clearance of the brain throughout the diurnal cycle and even in hypo- or hyper-glycemia [13]. Both hypo- and hyper-glycemic glucose concentrations in the medium of macrophages in vitro inhibit Aβ phagocytosis, but phagocytosis is restored in presence of PUFA in the medium. Diabetic patients have an increased risk of AD and may benefit from PUFA supplementation to provide missing energy in hypoglycemia and to enhance correct glycation and protect against glucose adducts in hyperglycemia of mild cognitive impairment (MCI) patients.

2.4 Monoclonal antibodies and PUFA in therapy of AD

The approval of aducanumab in 2021 increased enthusiasm for immune therapy by monoclonal antibodies. Aducanumab cleared both soluble oligomers and aggregated Aβ in a dose- and time-dependent fashion according to amyloid positron emission tomography (PET) imaging and slowed disease progression [18]. Both aducanumab and another monoclonal antibody ponezumab, however, have been associated with cerebro-vascular amyloid angiopathy (CAA) in models and with amyloid-related imaging abnormalities (ARIA) in patients [19]. Although the exact mechanisms of CAA related to aducanumab are not known due to the lack of pathological and biochemical studies, this complication appears to be due to macrophage shuttling Aβ from plaques to vessels, failure of macrophages in Aβ degradation and macrophage apoptosis with a release of Aβ into CAA vessels [10]. As PUFA up regulate the transcription of Aβ degradation enzymes, PUFA supplementation could be synergistic with the monoclonal antibody therapy.

3. Macrophages in amyotrophic lateral sclerosis

In amyotrophic lateral sclerosis (ALS), macrophages target aggregated superoxide dismutase (SOD-1) [20] and may be targeting free autologous DNA in the cytoplasm.

Immunopathological studies of sporadic amyotrophic lateral sclerosis (sALS) patients demonstrated an inflammatory attack by macrophages, cytotoxic T cells, NK and mast cells on motor neurons in the spinal cord [21, 22]. The attack includes gray matter and white matter of the spinal cord as well as peripheral nerves. The invading macrophages are inflammatory expressing cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), IL-6 and TNF-α, and they attack both caspase-positive and caspase-negative neurons. In addition, cytotoxic CD8 T cells and NK cells participate in the autoimmune attack expressing the cytotoxic enzymes granzymes.

Aggregated superoxide dismutase-1 (SOD-1) stimulates peripheral blood mononuclear cells (PBMC) of sALS patients inducing IL-1, IL6, and IL23. These cytokines enhance production of the auto-immune pro-inflammatory cytokine IL-17A, which is increased in the serum and spinal cord of ALS patients [23]. The induction of inflammatory pathology in sALS by SOD-1 in aggregated form is an example that in neurodegenerative diseases the aggregated state of the putative culprits SOD-1 in ALS and amyloid-β and P-tau in Alzheimer’s disease is sufficient for induction of inflammatory neuropathology.

4. Macrophages in HIV-1 infection

Trojan transport is the main path for virus penetration the blood-brain barrier in HIV-1 encephalitis. In addition, HIV-1 penetrates across coronary endothelia by transcytosis or by a paracellular route through disrupted tight junction protein ZO-1 [24].

Infiltration of target organs by inflammatory macrophages has a critical role in the progression of HIV-1 encephalitis and HIV-1 myocarditis. Other target organs in AIDS, the liver and the kidneys, are also infiltrated by macr0phages. In patients with HIV-1 encephalitis, cognitive impairment is proportionate to the number of macrophages infiltrating the brain [25]. In a study of postmortem heart tissues from 15 AIDS patients, the failing hearts showed significantly higher infiltration in the left ventricular myocardium by COX-2-positive and iNOS-positive macrophages compared to the functioning hearts. In the hearts with HIV-1 myocarditis, productive infection was exclusively in infiltrating macrophages and T cells, not in cardiomyocytes [26]. Significant infiltration of the failing heart by COX-2-positive macrophages (demonstrated using an antigen-retrieval technique) and production of inflammatory cytokines and mediators, such as NO, quinolinic acid, free radicals, is considered as the main mechanism of heart failure in HIV-1 myocarditis [26].

5. Macrophages in patients with COVID-19 infections

The recent epidemic of SARS-CoV-2 viral infection is associated with severe complications in subjects with risk factors, including older age, obesity, diabetes, renal failure; cancer; chronic obstructive pulmonary disease from smoking; and immunosuppressive conditions. In contrast, SARS-CoV-2 infected subjects without risk factors may suffer only mild pulmonary damage [27].

SARS-CoV-2 and previous coronaviruses responsible for severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS) induce inflammatory activation of monocytes/macrophages (MM). Patients with a low expression of interferon–α/β/γ in macrophages may suffer severe pulmonary damage by lung infiltration with inflammatory MM in a cytokine release syndrome (CRS), a.k.a. cytokine storm. MM are attracted into the lungs by chemokines produced by lung epithelia and fibroblasts. MM are activated by GM-CSF and IL-6, therefore, a blockade with IL-6 receptor antibody (tocilizumab, Actemra^R) is used in presence of a high inflammation [27]. CRS is related to the release of IL-1, IL-6 and nitric oxide (NO) from monocyte/macrophages, as shown in vitro and in an animal model by depletion of macrophages. CRS is similar to macrophage activation syndrome (MAS), a complication of several autoimmune diseases, in which well-differentiated macrophages show uncontrolled proliferation. CRS is also a complication of chimeric antigen receptor (CAR) therapy of B cell malignancies, in which macrophages produce IL-6, IL-1 and NO and where IL-1 blockade is therapeutic. COVID 19 pneumonia may be complicated by disseminated intravascular coagulation (DIC), therefore anticoagulation with enoxaparin (Lovenox^R) is routinely used in severe CoV2 infections.

In COVID-19 pneumonia, macrophages have a dual role with a beneficial anti-viral role and a detrimental role through excessive release of inflammatory cytokines [28]. The lung has two types of macrophages, interstitial and alveolar macrophages. The alveolar macrophages with M1 type are pivotal in defense against pathogens by phagocytosis of microbes, enhancement of development of cytotoxic T cells and type I interferon production. The M2 type alveolar macrophages dampen the inflammation and repair the damage in association with other immune tissue cells. Macrophages infected by CoV2, however, accumulate lipid droplets in association with virus replication [29].

Angiotensin-converting enzyme 2 (ACE2) is a cell receptor for SARS and CoV-2 on lung epithelial cells. SARS-CoV-2 can infect monocytes and macrophages through ACE2-dependent and ACE2-independent pathways [27]. A second study with similar findings detected ACE2-expressing CD68+ and CD169+ macrophages containing SARS-CoV-2 nucleoprotein antigens in the sinuses of the lymph nodes of COVID-19 patients. These infected macrophages up regulated the pro-inflammatory cytokines IL-6 [30] and IL-1B [31]. The increased production of these cytokines by infected monocytes and macrophages induces hyper-inflammation and the onset of cytokine storm, leading to excess tissue damage and the potential harm of other vital organs in addition to the lungs (Figure 2) [27]. In addition, a subset of macrophages isolated from Covid-19 patients have been found to express genes that promote fibrosis generation and tissue repair. Therefore, the infection of infiltrating macrophages might not only be detrimental because of the promotion of acute inflammation, but also because of fibrotic complications that may arise and that have been observed in patients under mechanical ventilation. Long-term increases in the pro-inflammatory M1 type macrophages are associated with elevated reactive oxygen species (ROS) [32], which can produce harmful long-term side effects.

Figure 2.

Monocyte infiltration of the lungs with differentiation and activation of macrophages with production of cytokines and chemokines. Created with BioRender.com.

6. Macrophages in tuberculosis

Similar to in COVID-19 infections, macrophages play a dual role in tuberculosis. Macrophages permissive for M. tuberculosis (Mtb) replication, they may control and eliminate the infection in the presence of appropriate innate and acquired responses [4].

M1 and M2 macrophages are distinguished by their activation type. M1 cells are activated by the T helper type 1 (Th1) cells. These cells pertain to cell mediated immune responses and emit interleukin-2 (IL-2), IFN-γ, and lymphotoxins [33]. Th1 cells lead to an increased M1 activation due to the production of IFN-y. Nitric oxide production is stimulated by the Th1 activation, rather than T helper type 2 (Th2) cells [34]. Th2 cells cause the activation of M2 macrophages. These cells are responsible for the stimulation of humoral immunity and secrete interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-10 (IL-10). Th2 responses are a result of the production of ornithine which activates the M2 response allowing for tissue repair [1]. In tuberculosis, the intersection of Th1 and Th2 activation of M1 and M2 respectively leads to the immunity against pathogens. Along with the production of IFN-y, immunoglobulin A (Ig-A) is necessary for defense against tuberculosis (Table 2) [35].

	Type 1 macrophages	Type 2 macrophages
Marker expression	CD80, CD54	CD163, CD206
Activation	Th1, INF-γ, GM-CSF	Th2, IL-4
Pro-inflammatory cytokines	High	Low
Antigen presentation	Yes	No
Production of ROS/NO	High	Low
Purpose	Destruction of microbes	Construct extracellular matrix

Table 2.

Type 1 and type 2 macrophage distinction.

Alveolar macrophages (AM) and dendritic cells phagocytize Mtb in the alveolar space in the lungs. Mtb is transmitted by macrophages to other organs via lymph nodes and blood vessels [36]. Alveolar macrophages present Mtb antigens to the adaptive immune cells to initiate an immune response. Subsequently, granulomas are formed because of the early inflammatory response caused by the AM [37]. Granulomas contain a variety of immune cells in order to contain the antigen. Some of the immune cells present in granulomas include foamy macrophages, epithelioid cells, neutrophils, and dendritic cells.

7. Conclusion

The phenotype of macrophages infiltrating the lesions in AD, ALS, HIV-1 myocarditis and encephalitis should be determined first, as it is the initial information necessary for analyzing macrophage role in each disease. M1 phenotype should be confirmed by staining of NOS, COX-2, TNF alpha, IL1, and IL-6 on macrophages.

Preliminary data suggest the following: (a) the pro-inflammatory M1 macrophage phenotype = CD54 + CD80/CD163 + CD206 > 4.0 is associated with an increased macrophage effector function in viral infections, but also with organ damage best known in HIV-1 myocarditis; b) the pro-resolution phenotype M1/M2 phenotype CD54 + CD80/CD163 + CD206 = in AD is associated with a beneficial role of macrophages in AD. The natural substances polyunsaturated fatty acids, vitamins B and D, energy molecules (carnitine), and flavonoids (resveratrol) may have individual health effects by improving macrophage transcriptome and macrophage pro-resolution type for brain clearance in AD, but the effects are complex and depend on many variables. In ALS patients, the M1/M2 phenotype promoted by fatty acids could be beneficial, but the data in human patients are not yet available.

Conflict of interest

The authors declare no conflict of interest.