Frontline Science: Targeted expression of a dominant-negative high mobility group A1 transgene improves outcome in sepsis

Abstract

High mobility group (HMG) proteins are a family of architectural transcription factors, with HMGA1 playing a role in the regulation of genes involved in promoting systemic inflammatory responses. We speculated that blocking HMGA1-mediated pathways might improve outcomes from sepsis. To investigate HMGA1 further, we developed genetically modified mice expressing a dominant negative (dn) form of HMGA1 targeted to the vasculature. In dnHMGA1 transgenic (Tg) mice, endogenous HMGA1 is present, but its function is decreased due to the mutant transgene. These mice allowed us to specifically study the importance of HMGA1 not only during a purely pro-inflammatory insult of endotoxemia, but also during microbial sepsis induced by implantation of a bacterial-laden fibrin clot into the peritoneum. We found that the dnHMGA1 transgene was only present in Tg and not wild-type (WT) littermate mice, and the mutant transgene was able to interact with transcription factors (such as NF-κB), but was not able to bind DNA. Tg mice exhibited a blunted hypotensive response to endotoxemia, and less mortality in microbial sepsis. Moreover, Tg mice had a reduced inflammatory response during sepsis, with decreased macrophage and neutrophil infiltration into tissues, which was associated with reduced expression of monocyte chemotactic protein-1 and macrophage inflammatory protein-2. Collectively, these data suggest that targeted expression of a dnHMGA1 transgene is able to improve outcomes in models of endotoxin exposure and microbial sepsis, in part by modulating the immune response and suggest a novel modifiable pathway to target therapeutics in sepsis.

Mesenchymal stromal cells expressing a dominant-negative high mobility group A1 transgene exhibit improved function during sepsis

“…qRT-PCR with SYBR Green Master Mix (Bio-Rad Laboratories, Hercules, CA) was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA) as described. 14,21 The samples were treated with DNase I to degrade any genomic DNA contamination, and cDNA synthesized by iScript TM Reverse Transcription Supermix (Bio-Rad Laboratories). qRT-PCR was performed for mouse HMGA1 using primers forward 5 ′ -GCTGGTCGGAGTCAGAAAG-3 ′ and reverse 5 ′ -GGTGACTTTCCGGGTCTTGG-3 ′ ; mouse cyclooxygenase-2 (Cox-2) using primers forward 5 ′ -GCCTACTACAAGTGTTTCTTTTTGCA-3 ′ and reverse 5 ′ -CATTTTGTTTGATTGTTCACACCAT-3 ′ ; mouse IL-6 using primers forward 5 ′ -ACAAGTCGGAGGCTTAATTACACAT-3 ′ and reverse 5 ′ -TTGCCATTGCACAACTCTTTTC-3 ′ ; mouse MIP-2 using primers forward 5 ′ -CCACCAACCACCAGGCTACAGGGGC-3 ′ and reverse 5 ′ -AGGCTCCTCCTTTCCAGGTCAGTTAGC-3 ′ , mouse KC using primers forward 5 ′ -ACCCGCTCGCTTCTCTGT-3 ′ and reverse 5 ′ -AAGGGAGCTTCAGGGTCAAG-3 ′ , and mouse stromal cellderived factor-1 (SDF-1) using primers forward 5 ′ -GAGCCAACGTCAAGCATCTG -3 ′ and reverse 5 ′ -CGGGTCAA TGCACACTTGTC-3 ′ .…”

“…The dnHMGA1 Tg mice were less hypotensive to endotoxemia and had lower mortality in bacterial sepsis. In addition, the Tg mice had reduced tissue infiltration of inflammatory cells during experimental sepsis 14 . As mesenchymal stromal cells (MSCs) are another mesenchymal cell with a significant influence on the inflammatory response during sepsis, 15–17 we expanded our appraisal of HMGA1 from vascular smooth muscle cells to MSCs.…”

“…12,13 To better understand the importance of HMGA1 in mesenchymal cells, we generated a transgenic (Tg) mouse overexpressing a dominant-negative (dn) form of HMGA1 targeted to mesenchymal cells of vascular smooth muscle origin. 14 This dn transgene is not capable of binding to DNA; however, it interacts with transcription factors. The dnHMGA1 Tg mice were less hypotensive to endotoxemia and had lower mortality in bacterial sepsis.…”

High Mobility Group A (HMGA): Chromatin Nodes Controlled by a Knotty miRNA Network

“…Therefore, establishing finely regulated control of HMGA expression is very important in the correct development and the maintenance of adult cellular homoeostasis. Accordingly, aberrant expression of HMGA proteins due to the dysregulation of their expression or the expression of mutated forms causes several diseases, such as different forms of neoplasia and metabolic disorders, which have been extensively reviewed [30][31][32], and it is involved in other pathologies such as polycystic ovary syndrome, sporadic Alzheimer's disease, myocardial infarction, obesity, ischaemia, atherosclerosis, and sepsis [24,28,[33][34][35][36][37]. Therefore, precise spatiotemporal regulation of the expression of HMGA factors is crucial in the correct development and preservation of adult physiological conditions.…”

Unhinging the machinery of sepsis: An unexpected role for vascular smooth muscle

“…In the current issue of The Journal of Leukocyte Biology , Baron et al. expand upon their prior findings and examine the role of HMGA1 in murine model of endotoxemia and Escherichia coli sepsis using a genetic approach . Because HMGA1 knockout mice develop cardiac abnormalities and hematologic malignancies, the authors designed a mouse that expresses a dominant negative form of HMGA1 (dnHMGA1) that inhibits HMGA1 biological activity in vascular smooth muscle cells .…”