Macrophages are essential components of the innate and adaptive immune systems and play central roles in inflammation and host defense. These cells are functionally classified into two major types: classically activated, proinflammatory (M1) macrophages and alternatively activated (M2) macrophages. M1 macrophages are induced by Th1 cytokines such as IFNγ and Granulocyte macrophage colony stimulating factor (GM-CSF) or lipopolysaccharide (LPS) and are characterized by cytotoxic activity against bacterial and viral infections and high expression levels of proinflammatory cytokines and chemokines. By contrast, M2 macrophages are induced by Th2 cytokines, such as IL-4, IL-13, IL-10, and TGF-βs, and they are characterized by efficient phagocytosis of dead cells and strong scavenger receptor expression with resolution of inflammation, tissue remodeling, fibrosis and tumor progression. In the acute inflammation phase during the early stage of tissue injury, neutrophils and monocytes heavily infiltrate the injured tissue from the blood to reach high M1/M2 ratios, and this is followed by the resolution of inflammation and the remodeling phase with an M2 macrophage-enriched environment. Several reports have indicated that the pathology of chronic inflammatory diseases, such as type 2 diabetes and atherosclerosis, and impaired healing is closely associated with the M1 and M2 macrophage balance. Especially in tissue repair, M2 macrophages may terminate tissue-destructive proinflammatory responses but create a reparative environment by cleaning up apoptotic dead cells and stimulating angiogenesis and cell proliferation. This event also seems to be an important step toward the acquisition of tolerance to self-antigens of apoptotic cells and avoidance of the induction of an autoimmune response, especially in IL-10-induced deactivating M2c-type macrophages. However, the origin and classification of these late-arriving, tissue-repairing M2 macrophages from the plastic transition of M1 macrophages, infiltration of newly generated M2-skewed monocytes or local proliferation of tissue macrophages in response to the Th2 cytokine IL-4, independently of monocytes, remain controversial. Although molecular signatures for M1/M2 macrophages have not yet been clearly resolved in human, mouse, and rat systems, the manipulation of M2 polarization could be a tempting pharmacological target for the treatment of chronic inflammation-associated metabolic disease and tissue repair.
Multiple intracellular signaling pathways, such as the JAK/STAT, PKC/ERK, and PI3K/Akt/mTOR pathways, function in parallel or convergently in M2 polarization of macrophages or monocytes under a variety of pathophysiological conditions. The Th2 cytokines IL-4 and IL-13 (IL-4/13) induce M2 polarization by activating STAT6, and these macrophages are defined as M2a subset. The anti-inflammatory cytokine IL-10 induces the activation of STAT3 and leads to the M2c subtype. Alternatively, activation of the PI3K/Akt/mTOR signaling pathway also leads to M2 polarization in steady-state macrophages or monocytes by skewing M1 macrophages to M2-type macrophages, and PI3K/Akt/mTOR inhibitors can prevent this M2 polarization of human macrophages and redirect their differentiation toward an M1 state. Bone morphogenic protein-7 (BMP-7) mediates monocyte polarization into M2 macrophages by activating SMAD/PI3K/Akt/mTOR. Recently, glucose metabolism and protein metabolism have been shown to regulate macrophage polarization, and the involvement of AMP-activated protein kinase (AMPK) α1 in M2 polarization has been noted in a muscle regeneration model. In addition, lipid metabolism is also involved in M2 polarization, as evidenced by the important mediator effects of PPAR family members in IL-4-induced M2 polarization. Additionally, the intracellular arginine balance seems to be an important regulator of M1/M2 polarization; nitric oxide (NO) is produced from arginine by inducible NO synthase (iNOS) in M1 macrophages, or ornithine is produced from arginine by Arginase-1 as a substrate for polyamines in M2 macrophages. Therefore, many intracellular signaling pathways and cellular metabolic states act together during M2 polarization.
SP, an undecapeptide, is a member of the tachykinin peptide family and acts as a sensory neurotransmitter and neuromodulator related to the nociceptive pain pathway in the central nervous system. SP has generally been known to activate immune cells into proinflammatory ones. However, in our previous study, SP treatment enhanced recovery from spinal cord injury in rats. As supporting evidence, a decrease in pro-inflammatory M1 markers such as iNOS and CD86, but an increase in the anti-inflammatory M2 markers Arginase-1 and CD206, was detected at an early stage in the lesion site of SP-treated SCI rats, which was also accompanied by reduced apoptosis of neurons and oligodendrocytes and stimulation of axon outgrowth. It has been proposed that SP may modulate injury-provoked acute inflammation at an early stage of SCI and thereby increase the proportions of CD11b+IL-10+CD206+ M2-like macrophages in the SCI microenvironment. Accordingly, we hypothesized that SP may play a role as a novel M2 cytokine to modulate bone marrow-derived monocytes or inflamed tissue macrophages to polarize M2 subtype macrophages, which may contribute positively to tissue repair and healing processes by resolving inflammation, cleaning up dead cells, and creating a more receptive microenvironment for tissue repair.
To test this hypothesis, we explored whether SP can directly induce M2 type macrophages from bone marrow-derived monocytes, GM-CSF-differentiated macrophages similar to inflammatory macrophages, and the THP-1 human monocytic cell line based on the expression of M1 and M2 subtype-specific markers such as Arginase-1, iNOS, CD163 scavenger receptor, CD206 mannose receptor, C
