Background Dendritic cells (DCs) are responsible for initiating adaptive immunity by interpreting environmental signals and then tailoring downstream immune responses to most effectively combat infections and malignancies. The cytokine AIMp1 is critical for promoting type 1 immunity and is associated with favorable long-term outcomes in cancers. Work from our group suggests DC-intrinsic AIMp1 is critical for DC maturation and ability to drive type 1 immune responses against influenza and melanoma. Further analysis identified that—possibly by regulating the phosphatase PP2A—AIMp1 specifically promotes phosphorylation of p38 MAPK downstream of toll-like receptor signaling. To build on that work here, we investigated the up- and down-stream signaling events involved in AIMp1-mediated p38 MAPK signaling regulation within DCs.

Methods We first generated bone marrow-derived dendritic cells from AIMp1 wild type (WT) and AIMp1 knockout (KO) mice. BMDCs from both groups were then stimulated with a pro-inflammatory cytokine cocktail (IL-1b, TNF, IL-6, PGE2) with or without toll-like receptor agonists lipopolysaccharide, staphylococcus enterotoxin B, or Poly(I:C). Western blotting was used to evaluate differences in total protein expression and/or phosphorylation of the major PP2A inhibitors ANP32A & Protein SET1 upstream of p38 MAPK as well as the AP-1 Transcription factor subunits c-Fos and c-Jun downstream of p38 MAPK.

Results Regardless of the stimulation conditions, AIMp1 KO DCs demonstrated increased expression of ANP32A. Meanwhile, the total expression of SET1 was unchanged, but it’s 20kDa cleavage product was increased in AIMp1 KO BMDCs. Similarly, we observed increased phosphorylation of both c-Fos and c-Jun in AIMp1 KO BMDCs regardless of the stimulation conditions.

Conclusions Given that p38 MAPK is a major regulator of AP-1 transcription factors, the results indicate that dysregulation of p38 MAPK following the loss of AIMp1 may cause dysregulate in AP-1 transcription factor subunit phosphorylation, dimerization, and function. AP-1 dysregulation may in turn have significant impacts on the function of DCs since they can regulate the production of critical cytokines, such as IL-12p70, that are important drivers of type 1 immune responses. Moreover, the dysregulation ANP32A and SET1 cleavage product supports previous observations that AIMp1 indirectly regulates p38 MAPK through PP2A since they are the two major non-redundant regulators of PP2A. Altogether, this work underscores the importance of AIMp1 in immune polarization and offers potential molecular targets for modulating DC function in disease.

Ethics Approval The work outlined in this abstract as conducted with approval from the Baylor College of Medicine IACUC in accordance with the ethical treatment of research animals (Protocol AN-1428).