IL-1β-Induced Inflammatory Transcriptome and TGF-β1 Attenuation of Chemokine Expression in ACL Remnant Fibroblasts.

BACKGROUND: The high incidence of posttraumatic osteoarthritis after anterior cruciate ligament (ACL) injury and reconstruction suggests that biological mechanisms beyond joint instability contribute to disease progression. After injury, ACL-resident cells are exposed to inflammatory mediators within the joint and may influence the local inflammatory microenvironment; however, their global transcriptomic responses to inflammatory stimuli remain underexplored.

HYPOTHESIS: Interleukin-1 beta (IL-1β) induces inflammatory transcriptomic responses in ACL remnant-derived (ACLr) fibroblasts, and transforming growth factor beta 1 (TGF-β1) attenuates these responses.

METHODS: Primary ACLr fibroblasts were isolated from ACL remnants (n = 10) collected at the time of knee surgery. Near-confluent cultures were treated with IL-1β (10 ng/mL) for 24 hours to model an inflammatory joint environment. Genome-wide transcriptional changes were assessed by RNA sequencing using an Illumina NovaSeq-6000, with selected targets confirmed by microfluidic digital polymerase chain reaction (PCR). To evaluate the effects of TGF-β1, ACLr fibroblasts pretreated with IL-1β were exposed to TGF-β1 (10 ng/mL) for 48 hours, and chemokine transcript levels were assessed using digital PCR.

RESULTS: IL-1β induced broad inflammatory transcriptional responses characterized by upregulation of chemokines (CXCL6, CXCL8, CXCL5, CCL20, CXCL1, and CXCL3), cytokines (IL33, IL1B, and IL6), and matrix-degrading enzymes (MMP3 and MMP12), while suppressing extracellular matrix-associated genes (KRT14, COL21A1, POSTN, COL1A1, and COL6A3). Functional enrichment analysis demonstrated activation of immune and inflammatory responses, cytokine and chemokine signaling, and cell activation. TGF-β1 reduced the expression of all assessed IL-1β-induced chemokines, with reduction of up to 50%, with statistically significant suppression observed for 13 of the 16 chemokines. Concomitantly, TGF-β1 treatment reduced expression of CEBPB, NFKBIA, and NFKBIZ, transcripts implicated in inflammatory gene regulation.

CONCLUSION: ACLr fibroblasts mounted a robust inflammatory transcriptomic response to IL-1β, characterized by marked induction of chemokines and inflammatory pathways. TGF-β1 attenuated many of these responses, supporting its role as a regulator of inflammatory signaling in ACL-resident cells.

CLINICAL RELEVANCE: ACL-resident cells respond to injury and may contribute to the post-injury inflammatory microenvironment. Therapeutic modulation of inflammatory signaling in these cells could represent a strategy to mitigate early molecular events associated with post-traumatic osteoarthritis following ACL tear.

STUDY DESIGN: Descriptive laboratory study.