Macrophage migration inhibitory factor activates inflammatory responses of astrocytes through interaction with CD74 receptor

Astrocytes have been shown to produce several pro- and anti-inflammatory cytokines to maintain homeostasis of microenvironment in response to vast array of CNS insults. Some inflammation-related cytokines are responsible for regulating such cell events. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that can be inducibly expressed in the lesioned spinal cord. Unknown is whether MIF can facilitate the production of immunosuppressive factors from astrocytes to tune milieu following spinal cord injury. Following establishment of contusion SCI rat model, correlation of PGE2 synthesis-related protein levels with that of MIF was assayed by Western blot. ELISA assay was used to detect production of PGE2, TNF-α, IL-1β, and IL-6. Immunohistochemistry was performed to observe colocalization of COX2 with GFAP- and S100β-positive astrocytes. The primary astrocytes were treated by various inhibitors to validate relevant signal pathway. The protein levels of MIF and COX2, but not of COX1, synchronously increased following spinal cord injury. Treatment of MIF inhibitor 4-IPP to the lesion sites significantly reduced the expression of COX2, mPGES-1, and as a consequence, the production of PGE2. Astrocytes responded robustly to the MIF interference, by which regulated MAPK/COX2/PGE2 signal pathway through coupling with the CD74 membrane receptor. MIF-induced production of PGE2 from astrocytes was able to suppress production of TNF-α, but boosted production of IL-1β and IL-6 in LPS-activated macrophages. Collectively, these results reveal a novel function of MIF-mediated astrocytes, which fine-tune inflammatory microenvironment to maintain homeostasis. These suggest an alternative therapeutic strategy for CNS inflammation.

Astrocytes act as immune effector cells with the ability to produce a wide array of chemokines and cytokines in response to various stimuli. Macrophage migration inhibitory factor (MIF) is inducibly expressed in injured spinal cord contributing to excessive inflammation that affects motor functional recovery. Unknown is whether MIF can facilitate inflammatory responses through stimulating release of chemokines from astrocytes following spinal cord injury. Following the establishment of the contusion spinal cord injury rat model, the correlation of chemokine (C-C motif) ligand 5 (CCL5) expression with that of MIF was assayed by Western blot, ELISA, and immunohistochemistry. Immunoprecipitation was used to detect MIF interaction with membrane CD74 receptor. Intracellular signal transduction of MIF/CD74 axis was analyzed by transcriptome sequencing of primary astrocytes and further validated by treatment of various inhibitors. The effects of CCL5 released by astrocytes on macrophage migration were performed by transwell migration assay. The post-injury locomotor functions were assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. The protein levels of chemokine CCL5/RANTES were remarkably increased in the astrocytes of rat injured spinal cord, in parallel with the expression of MIF. Treatment of MIF inhibitor 4-IPP in the lesion sites resulted in a significant decrease of CCL5 protein levels. In vitro study revealed MIF was capable of facilitating CCL5 production of astrocytes through interaction with CD74 membrane receptor, and knockdown of this receptor attenuated such effects. Production of CCL5 in astrocytes was significantly blocked by inhibitor of c-Jun N-terminal kinase, rather than by those of ERK and P38. Recombinant CCL5 protein was found to be more effective in promoting migration of M2- compared to M1-type macrophages. Collectively, these data reveal a novel function of MIF in regulation of CCL5 release from astrocytes, which in turn favors for recruitment of inflammatory cells to the injured site of the spinal cord, in association with activation of excessive inflammation.

Background: Astrocytes are the predominant glial cell type in the central nervous system (CNS) that can secrete various cytokines and chemokines mediating neuropathology in response to danger signals. D-dopachrome tautomerase (D-DT), a newly described cytokine and a close homolog of macrophage migration inhibitory factor (MIF) protein, has been revealed to share an overlapping function with MIF in some ways. However, its cellular distribution pattern and mediated astrocyte neuropathological function in the CNS remain unclear. Methods: A contusion model of the rat spinal cord was established. The protein levels of D-DT and PGE₂ synthesis-related proteinase were assayed by Western blot and immunohistochemistry. Primary astrocytes were stimulated by different concentrations of D-DT in the presence or absence of various inhibitors to examine relevant signal pathways. The post-injury locomotor functions were assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. Results: D-DT was inducibly expressed within astrocytes and neurons, rather than in microglia following spinal cord contusion. D-DT was able to activate the COX2/PGE₂ signal pathway of astrocytes through CD74 receptor, and the intracellular activation of mitogen-activated protein kinases (MAPKs) was involved in the regulation of D-DT action. The selective inhibitor of D-DT was efficient in attenuating D-DT-induced astrocyte production of PGE₂ following spinal cord injury, which contributed to the improvement of locomotor functions. Conclusion: Collectively, these data reveal a novel inflammatory activator of astrocytes following spinal cord injury, which might be beneficial for the development of anti-inflammation drug in neuropathological CNS.

JOURNAL/nrgr/04.03/01300535-202306000-00032/inline-graphic1/v/2022-11-21T174919Z/r/image-tiff