Ahad Azimuddin

 Introduction. Chronic diabetic wounds (CDWs) present a significant physical and financial burden on our society. More than 6.5 million Americans suffer from non-healing wounds, costing $35.3 billion to Medicare.¹ Pathologic wound repair disrupts any of the four stages of physiologic wound repair: hemostasis, inflammation, proliferation/repair, and remodeling.^1,2 In the inflammation stage, CDWs lack proper macrophage polarization from the pro-inflammatory (CX₃CR1^loLy6^hi) to the anti-inflammatory (CX₃CR1^hiLy6^low) macrophage.^3,4 The many mechanisms by which this polarization is controlled have yet to be elucidated. However, recent studies find that Setdb2, a histone methyltransferase, plays a role in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways dictating macrophage polarity.^5,6,7 These findings suggest Setdb2 enhancement as a promising approach to restoring proper inflammation in CDWs. Methods. Setdb2^LacZ reporter mice were infected with Influenza A Virus (IAV) to quantify Setdb2 expression and inflammatory cytokine production. Leukocytes were measured by various surface markers.⁶ Separately, tamoxifen-induced inhibition of NF-KB in skeletal stem cells was used to view the impact of NF-KB on diabetic inflammation.⁷ Finally, control mice were compared with Diabetic Induced Obese (DIO) mice and analyzed for inflammatory cytokine expression.⁵ Results. IAV induced Ifnb1 expression lead to an 11-fold increase in Setdb2 transcript compared to uninfected lungs (11.0 ± 1.38 vs. 1.0 ± 0.43; p<0.001). Pro-inflammatory cytokine ligands Ccl2 and Ccl5, respectively, were upregulated 23- and 29-fold in Setdb2^-/- in bone marrow-derived macrophages.⁶ This result validates association between Setdb2 expression and the consequential inflammatory cytokine production. Additionally, NF-κB aberrant activation was shown in diabetic wild-type mice, leading to a release of pro-inflammatory cytokines that support CX₃CR1^loLy6^hi.⁷ The diabetic increase of Tnf, Il1b, and Ifng was subsequently decreased to normal levels with the inhibition of NF-κB.⁷ The reversal of aberrant NF-κB expression restored normal inflammatory cell level, indicating NF-κB plays a role in wound repair dysregulation.⁷ Finally, Setdb2 was found to be directly responsible for increased inflammatory cytokines in DIO mice. Five-day post-injury DIO mice macrophages had reduced Setdb2 histone trimethylation at the NF-κB binding site on IL1β, TNFα, and NOS2 promoters compared to non-diabetic control mice.⁵ This result confirms the relationship between Setdb2, NF-κB, and the production of inflammatory cytokines that inhibit the necessary transformation of CX₃CR1^loLy6^hi to CX₃CR1^hiLy6^low in diabetic patients. Conclusions. Setdb2 inhibition of NF-κB promotes macrophage polarization necessary for healing, but its expression in CDWs is pathologically reduced. Therefore, targeted restoration of Setdb2 could be a viable strategy in improving anti-inflammatory macrophage function.

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