Nucleus pulposus cells regulate macrophages in degenerated intervertebral discs via the integrated stress response-mediated CCL2/7-CCR2 signaling pathway

Lower back pain (LBP), which is a primary cause of disability, is largely attributed to intervertebral disc degeneration (IDD). Macrophages (MΦs) in degenerated intervertebral discs (IVDs) form a chronic inflammatory microenvironment, but how MΦs are recruited to degenerative segments and transform into a proinflammatory phenotype remains unclear. We evaluated chemokine expression in degenerated nucleus pulposus cells (NPCs) to clarify the role of NPCs in the establishment of an inflammatory microenvironment in IDD and explored the mechanisms. We found that the production of C-C motif chemokine ligand 2 (CCL2) and C-C motif chemokine ligand 7 (CCL7) was significantly increased in NPCs under inflammatory conditions, and blocking CCL2/7 and their receptor, C-C chemokine receptor type 2(CCR2), inhibited the inductive effects of NPCs on MΦ infiltration and proinflammatory polarization. Moreover, activation of the integrated stress response (ISR) was obvious in IDD, and ISR inhibition reduced the production of CCL2/7 in NPCs. Further investigation revealed that activating Transcription Factor 3 (ATF3) responded to ISR activation, and ChIP-qPCR verified the DNA-binding activity of ATF3 on CCL2/7 promoters. In addition, we found that Toll-like receptor 4 (TLR4) inhibition modulated ISR activation, and TLR4 regulated the accumulation of mitochondrial reactive oxygen species (mtROS) and double-stranded RNA (dsRNA). Downregulating the level of mtROS reduced the amount of dsRNA and ISR activation. Deactivating the ISR or blocking CCL2/7 release alleviated inflammation and the progression of IDD in vivo. Moreover, MΦ infiltration and IDD were inhibited in CCR2-knockout mice. In conclusion, this study highlights the critical role of TLR4/mtROS/dsRNA axis-mediated ISR activation in the production of CCL2/7 and the progression of IDD, which provides promising therapeutic strategies for discogenic LBP.

The first numAuxModelSamples are used to train the auxiliary model parameters.5000000 The first numPreAuxModelSamples will have their assignment likelihoods and contributions to the transcript abundances computed without applying any auxiliary models.Other materials and methods

ELISA
The levels of CCL2 and CCL7 in the supernatant of NPCs were detected by CCL2/7 ELISA kits (Ruixin Biotech, China).All processes followed the manufacturer's standard operating procedures.The absorbance was detected by an F50 enzyme labeling instrument (TECAN, Switzerland) and converted into content by the standard curve method.

Hematoxylin and eosin (HE) and Safranin O-Fast Green (SO) staining
For HE staining, dewaxed paraffin sections were stained with hematoxylin solution and then differentiated with 8% hydrochloric acid-alcohol differentiation solution at 25 °C for 2 s.The sections were then treated with bluing buffer for 4 min and washed in 95% alcohol.Next, the sections were soaked in eosin dye for 2 s and washed with anhydrous alcohol.For SO staining, the dewaxed paraffin sections were stained with fresh Weigert solution for 5 mins and then differentiated by 8% hydrochloric acid-alcohol differentiation solution at 25℃ for 15 s.The sections were then stained for 5 mins with fast green solution and washed with pure water until the cartilage tissue was colorless.Next, safranin O solution was used to stain until the section cartilage was bright red.Finally, the stained sections were blocked with neutral gum and visualized by an Olympus BX51 microscope (Olympus, Japan).

Table 3. siRNA sequences siRNA Type Sequence
If this option is enabled, then no (lower) threshold will be set on how short bias correction can make effecctive lengths.
FalseNumber of fragment mappings to use when learning the sequence-specific bias model.2000000

Table 5 . Gene markers used for identification of macrophage subpopulations with scType
5000Use the traditional EM algorithm for optimization in the batch passes.