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Neuroinflammation, defined as an inflammatory response within the central nervous system (CNS), is a process triggered by different factors, including infection (e.g. COVID-19, toxoplasmosis, malaria), toxic metabolites, trauma, bleeding or autoimmunity processes (Tohidpour et al., 2017). Inflammation in the CNS occurs as a result of cells sensing pathogen-associated molecular patterns (PAMPs, e.g., Lipopolysaccharide (LPS) or lipoteichoic acid (LTA) from bacteria) or damage-associated molecular patterns (DAMPs, e.g., nuclear or cytosolic proteins or nucleic acids from damaged neurons, extracellular ATP or parasite hemozoin) via pattern-recognition receptors (PRRs).
Neuroinflammation is considered neuroprotective in its normally innate response when the inflammatory activity is for a shorter period; however, prolonged or maladaptive neuroinflammation is a key pathological driver of many neurological diseases, including neurodegenerative (e.g., Alzheimer’s and Parkinson’s diseases, epilepsy and motor degenerative disease) and psychiatric diseases (like schizophrenia and depression), as well as stroke, brain cancer, pain syndromes and traumatic brain injury (Chaney et al., 2021).
Immune activation in the CNS involves microglia and astrocytes, as well as endothelial and perivascular cells. It triggers a transient, self-limiting immune system response and initiates tissue repair. When the normal resolution mechanism fails, persistent inflammatory responses develop that are detrimental and inhibit regeneration. Persistent inflammation can be triggered by endogenous (e.g., genetic mutation, protein aggregates such as α-synuclein or amyloid β) or environmental (e.g., infection, trauma) factors. An unhealthy lifestyle, obesity, diabetes, mental stress, sleep loss and aging also increase the likelihood of neuroinflammation.
Microglia, astrocytes, and oligodendrocytes, the glial cells ubiquitously distributed within the brain parenchyma, together with brain-associated macrophages (BAMs) contribute in a coordinated manner to initiate and modulate the brain's inflammatory response.
Previously, it was believed that macrophages, microglia, and astrocytes underwent a simplistic polarization into either a pro-inflammatory or anti-inflammatory phenotype. These phenotypes were referred to as M1/M2 for microglia and macrophages (The M1 and M2 Paradigm of Macrophage and Microglia Polarization), or A1/A2 for astrocytes. According to this view, immune cells in the pro-inflammatory phenotype would initiate inflammation by releasing pro-inflammatory cytokines, recruiting other immune cells, and inducing inflammatory responses in neighboring cells. Conversely, cells in the anti-inflammatory phenotype would regulate and potentially halt the inflammatory process by releasing anti-inflammatory cytokines and promoting tissue repair through the release of trophic factors.
However, recent research, especially through genome-wide expression profiling studies, has shed light on the complexity of these cell phenotypes. It has been revealed that these cells can undergo a spectrum of phenotypic states, indicating a more nuanced and finely regulated neuroinflammatory process than previously expected. These new findings challenge the simplistic view of polarization and highlight the multifaceted nature of these cell types during inflammation.
Microglia are the resident innate immune cells of the CNS and account for approximately 10% of the cells in the CNS (Webers et al., 2020) and 20% of the glial cell population of the brain (Troncoso-Escudero et al., 2018). Microglial cells are characterized by highly dynamic processes, which aid in the detection of pathogens and subtle changes in the microenvironment when they are at rest. They act as sentinels that monitor the brain via surface receptors that recognize complement fragments, immunoglobulins, adhesion molecules, chemokine receptors (CCR), toll-like receptors (TLR), purinoreceptors, and scavenger and Fc receptors (Jurga et al., 2020; Prinz et al., 2021; Dermitzakis et al., 2023).
The resting state is transformed into an activated state by danger signals, including PAMPs and DAMPs. Activated microglia communicate by releasing cytokines, which alert surrounding cells and affect their function. They produce both proinflammatory (e.g., tumor necrosis factor-alpha (TNF-α), interleukins IL-1β, IL-6, IL-12, and IL-18, interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS) and monocyte chemotactic protein 1 (MCP-1)), as well as anti-inflammatory mediators (e.g., interleukins IL-4, IL-10 and IL-13, and transforming growth factor-beta (TGF-β)) (Angiulli et al., 2021). Through the release of these cytokines, microglia cells help to recruit other immune cells to the site and initiate the immune defence against pathogens or promote tissue repair.
Figure 2: P2Y12 is expressed in resting microglia. Indirect immunostaining of PFA fixed mouse cortex section with mouse anti-P2Y12 (Cat. 476 011, 1:2000, red) and guinea pig anti-IBA1 (cat. no. 234 308, 1:500, green). Nuclei were stained with DAPI (blue).
Figure 3: Indirect immunostaining of (A): PFA fixed wild-type and (B): triple transgenic Alzheimer’s disease mouse brain cortex sections with guinea pig anti-CD11c (cat. no. HS-375 004, dilution 1:1000; red) and rabbit anti-IBA1 (cat. no. HS-234 013, dilution 1:1000; green). The red signal shows CD11c expression only in activated microglia. Nuclei were stained with DAPI (blue).
Remarkably, activation of microglia is accompanied by changes in protein expression. Proteins characteristic of resting microglia, such as P2Y12 (Figure 2), CX3CR1, HexB, or TMEM119, are downregulated, whereas other proteins, such as CD11c (Figure 3), CD86 (Figure 4), CD11b, IBA1, CD68, F4/80 (mouse specific) and Galectin-3 (Figure 5), are upregulated or expressed only upon microglial activation (Benmamar-Badel et al., 2020, Jurga et al., 2020, Almolda et al., 2015, Garcia-Revilla et al., 2022).
In addition to these acute changes, microglia exhibit a long-lasting change after exposure to inflammatory stimuli and become more sensitive to potentially milder stimuli. On the one hand, microglial priming prepares microglia cells for later immune changes; on the other hand, constant exposure of microglia to infections throughout life may contribute to neurodegenerative diseases (Lima et al., 2022).
Microglial transcriptome changes have been identified in aged brain leading to enhanced inflammation, impaired phagocytosis, and profound morphological changes that reduce immune surveillance. In mouse models of neurodegenerative diseases, microglia cells with a “disease-associated microglial” (DAM) profile have been described (Finger et al., 2022).
Figure 4: (A): Activated microglia in the ipsilateral hippocampus of a mouse stroke brain highly express CD86 (green) and CD11b (red). (B): Higher magnification of the hippocampal region reveals co-localization of both markers in a subset of activated microglia. Indirect immunostaining of a formalin fixed paraffin embedded mouse stroke brain section (14 days after middle cerebral artery occlusion (MCAO) using Tyramide signal amplification with rabbit anti-CD86 (cat. no. HS-466 003, 1:600, green) and rat anti-CD11b (cat. no. HS-384 117, 1:250, red). Nuclei were stained with DAPI (blue).
Figure 5: Activated microglia in the lesion area of a mouse stroke brain highly express Galectin-3 (pink), CD11b (red) and IBA1 (green). Indirect immunostaining of PFA fixed mouse brain section (14 days after MCAO) with (A): rat anti-Galectin-3 (cat. no. HS-477 017, 1:500 pink), (B): rabbit anti-CD11b (cat. no. HS-384 008, 1:500, red) and (C): guinea pig anti-IBA1 (cat. no. 234 308, 1:500, green). (D): Higher magnification of the lesion area reveals different microglial phenotypes expressing either IBA1 alone or in combination with CD11b and/or Galectin-3. Nuclei were stained with DAPI (blue).
