CD74: a prospective marker for reactive microglia?

Cluster of differentiation 74 (CD74), also called as major histocompatibility complex class II (MHCII) invariant chain, is involved in trafficking MHCII cell surface molecules on antigen-presenting cells and has been implicated in many signaling pathways. For example, the interaction between CD74 and macrophage migration inhibitory factor cytokine (MIF) leads to the activation of a plethora of pathways such as extracellular regulated protein kinases, phosphoinositide 3-kinase, and nuclear factor-κB which are essential for cell survival, differentiation, and proliferation. Structurally, CD74 is a type 2 transmembrane receptor with a short N-terminal cytoplasmic tail consisting of 28 amino acids, a 24-amino acid transmembrane region, and a luminal domain of approximately 150 amino acids. In mice, Cd74 occurs in two isoforms, namely p31, and p41, due to alternative splicing. In humans, along with the p33 and p41 isoforms that correspond to the p31 and p41 isoforms of mice, two additional isoforms p35 and p43 can also be found (Farr et al., 2020). Microglia are central nervous system (CNS)-specific antigen-presenting cells with diverse functional repertoire. Microglia remain in a resting, albeit a surveying state under normal physiological conditions. Such microglia are deemed to be in their homeostatic state with a characteristic expression of Tmem119, Olfml3, P2ry12, Sall1, Hexb, Gpr34, or Fcrls. They however become reactive under pathological states assuming phenotypes with increased expression of genes such as ApoE, Axl, Clec7a, Cst7, Cybb, and Ctsb. It is now widely accepted that this microglial activation and the resultant dysregulation is an inevitable component of almost all CNS pathologies (Pettas et al., 2022).

Several studies have shown that MHCII-related molecules are upregulated in neurodegenerative conditions such as Alzheimer’s disease (AD). CD74 traffics these molecules from the endoplasmic reticulum to the cell surface, where it acts as a receptor for the pro-inflammatory molecule MIF. As such, the role of CD74 in various CNS diseases from the standpoint of microglia has been under keen investigation (Bryan et al., 2008). Single-cell sequencing techniques have provided important information regarding the role of CD74 during neurodegenerative conditions. scRNA-Seq of microglia from 5xFAD mice showed an increased Cd74 expression in the newly described AD-associated phenotype of microglia called disease-associated microglia. Cells in the cluster with Cd74 showed reduced homeostatic marker expression and increased levels of various other genes such as Itgax, Ccl6, and Apoe, etc. which are considered as activation markers of microglia (Keren-Shaul et al., 2017). Mathys et al. (2019) analyzed 89,660 single-nucleus transcriptomics from 48 AD patients. They identified a microglia subpopulation with increased expression of CD74 and other MHCII-related genes. Interestingly, this subpopulation also showed a significant overlap with the mouse disease-associated microglia cluster reported by Keren-Shaul et al. (2017). Another human microglia single-cell analysis identified a unique “activated response microglia” in response to amyloid-β plaques. This cluster was also enriched for inflammatory genes as well as MHCII genes where CD74 was significantly overexpressed (Sala Frigerio et al., 2019). These findings suggest a possible role for CD74 in understanding the role of microglia-mediated neuroinflammation in AD pathology in both humans and mouse models. CD74 also seems to be involved in the pathology of multiple sclerosis. Single-cell studies in humans revealed an upregulation of CD74 in multiple sclerosis-associated microglial clusters (Masuda et al., 2019). Moreover, CD74⁺ microglia were observed in the hippocampal CA1 region, 5 days after ischemic insults suggesting an important role of microglia CD74 signaling in neuronal death which is a hallmark ischemic brain injury (Hwang et al., 2017). Accumulating evidence suggests that aging of the CNS is associated with whole-brain inflammation and microglia in these brains are considered to be in a “primed state”. Single-cell sequencing of aged microglia revealed a unique microglial cluster with increased Cd74 expression. Mechanistically, it is reported that MIF released by neurons acts on microglia CD74 resulting in reactive microglia states associated with aging (Jin et al., 2022)

In vitro, CD74 levels were reported to be important in regulating interferon gamma (IFN-γ) signaling. Microglia react by increasing IFN-γ expression upon MIF treatment. It has also been reported that silencing CD74 signaling in microglia results in increased IFN-γ levels. Furthermore, it was shown that the treatment of cultured human microglia with IFN-γ increased CD74 expression. These findings suggest a possible feedback mechanism between MIF-CD74 and IFN-γ (Peferoen et al., 2015). MIF binding also leads to cleavage and release of the cytosolic intracellular domain of CD74. Resultant intracellular domain of CD74 interacts with transcription factors such as Runt-related transcription factor (RUNX) and nuclear factor-κB to bind to regulatory regions of genes implicated in apoptosis, immune responses, and immune regulation. Additionally, recent studies have shown that the treatment of murine microglial cell line BV2 as well as primary microglia with LPS leads to an increase in CD74 levels (Jahn et al., 2022).

Owing to the pro-inflammatory functions of CD74 and its strong activity in disease-associated and reactive microglia, identifying the factors that can regulate the expression of CD74 has become important. Transforming growth factor β (TGFβ) signaling in microglia is essential for the induction of homeostatic and maturation signatures in microglia. Our recent research found that TGFβ1 counteracts the LPS-induced increase in Cd74 expression in microglia. In addition, inhibiting TGFβ signaling in vitro with a TGFβ receptor type I inhibitor or in vivo by targeting microglia-specific Tgfbr2 has also been shown to result in a significant increase in Cd74 expression, further supporting the idea that CD74 is a marker for reactive microglia (Zöller et al., 2018; Jahn et al., 2022). Together, these results raise an interesting question about the functions of CD74 in microglia, which could be explored by targeting Cd74 expression specifically in microglia.

As mentioned above, human CD74 has four isoforms (p33, p41, p35, and p43), while murine CD74 has two isoforms (p31 and p41). However, so far, few studies have addressed the functional relevance of these isoforms in microglia. This indeed forms a very interesting prospect for future studies. In our recent study, we have demonstrated that LPS treatment results in an increase of p41 isoform levels, while blocking TGFβ signaling leads to p31 isoform upregulation (Jahn et al., 2022). These changes suggest isoform-specific roles in different signaling pathways and provide a foundation for further research into the role of CD74 isoforms in neurodegenerative diseases which can be addressed using isoform-specific targeting in microglia.

In summary, CD74 is being recognized as a marker for reactive microglia due to its increased expression levels under disease conditions compared to healthy CNS (Figure 1). However, the classification of CD74 as such should be done with caution as not much is known about its functions. Moreover, microglia phenotypes and so-called activation states are not a black-and-white scenario but rather exist as an outcome of multi-level regulation. Further studies are needed that take these factors into account to fully understand the functional role of CD74 in microglia reactivity.

Figure 1.

CD74 in homeostatic and reactive microglia.

Under homeostatic conditions, microglia cells are characterized by low levels of CD74 and high levels of P2ry12, Tmem119, and Hexb. However, certain stimuli such as LPS, interferon-γ, and the loss of signaling mechanisms like TGFβ1, as well as neurodegenerative conditions such as Alzheimer’s disease, multiple sclerosis, and cancer, can cause microglia to shift to a “reactive” phenotype. In this state, they display higher levels of Cd74 and reduced homeostatic markers. Aging and dying neurons can also activate CD74 by releasing its ligand MIF, stimulating pro-inflammatory pathways and leading to a reactive microglial phenotype. More research is needed to understand the role of microglia-specific CD74 and its isoforms. This could be achieved through the use of various approaches such as epigenetics, transcriptomics, metabolomics, and proteomics to study how microglial CD74 is targeted to regulate their behavior. AD: Alzheimer’s disease; CD74: cluster of differentiation 74; EAE: experimental autoimmune encephalomyelitis; Hexb: hexosaminidase subunit beta; IFNg: interferon-γ; LPS: lipopolysaccharide; MIF: macrophage migration inhibitory factor; NF-κB: nuclear factor κ-light-chain-enhancer of activated B cells; P2ry12: purinergic receptor P2Y12; TGFβ1: transforming growth factor β1; TgfβR2: transforming growth factor beta receptor 2; Tmem119: transmembrane protein 119. Created with BioRender.com.

Additional file: Open peer review report 1 ^{(77.5KB, pdf)} .