Cell Study Reveals How TRIM28 Fires Up Inflammatory Macrophages to Drive Keratinocyte Overgrowth

A single immune-regulating protein may act as a hidden switch that transforms quiet macrophages into inflammation-fueling cells capable of pushing skin keratinocytes into overdrive. In a carefully controlled laboratory study published in Immunity, Inflammation and Disease, researchers report that the protein TRIM28 promotes pro-inflammatory macrophage polarization and, through the chemical signals those macrophages release, drives keratinocyte proliferation and invasion. The work was conducted entirely in an in vitro co-culture system, meaning the findings describe a cellular mechanism rather than a confirmed disease process, but the mechanistic clarity of the study has drawn attention from immunologists interested in inflammatory skin conditions such as psoriasis.

The research team, led by Zhe Gao and Xin Zhang, set out to explore a question that has remained surprisingly open: what role, if any, does TRIM28 play in regulating the behavior of macrophages and the keratinocytes they communicate with? TRIM28, short for tripartite motif-containing 28, is a multifunctional protein best known for its involvement in transcriptional regulation and epigenetic modification. It can influence protein stability through ubiquitination and SUMOylation, two post-translational modifications that attach small protein tags to targets and alter their behavior. Despite growing interest in TRIM family proteins across immunology and cancer biology, TRIM28’s role in macrophage polarization had not been clearly defined.

To probe this question, the researchers used a well-established laboratory model. They cultured human THP-1 monocytic cells and differentiated them into macrophage-like cells using a chemical called phorbol 12-myristate 13-acetate. Separately, they maintained HaCaT keratinocytes, an immortalized human skin cell line. Using small interfering RNAs, they silenced TRIM28 expression in the macrophages, and in complementary experiments they boosted TRIM28 levels using an overexpression plasmid. These two approaches, gene knockdown and gene overexpression, allowed the team to observe what happens when TRIM28 is removed or amplified within the same cellular environment.

The results were striking. When TRIM28 was silenced, flow cytometry analysis revealed a marked shift in macrophage phenotype: the proportion of CD68-positive CD206-positive cells, associated with an M2-like or anti-inflammatory profile, increased to 25 percent from lower baseline levels. Conversely, the proportion of CD68-positive CD86-positive cells, associated with an M1-like or pro-inflammatory profile, dropped to just 5 percent. Enzyme-linked immunosorbent assays confirmed the functional consequences: silencing TRIM28 reduced secretion of the pro-inflammatory cytokines IL-6 and TNF-α while increasing the anti-inflammatory cytokine IL-10. Western blotting reinforced these findings, showing decreased expression of the M1-associated marker iNOS and increased expression of the M2-associated marker Arg1. When TRIM28 was overexpressed, every one of these trends reversed, pushing macrophages toward the pro-inflammatory state.

The team then traced the molecular pathway underlying this effect. Prior research had suggested that TRIM28 can stabilize NLRP3, a key component of the inflammasome, a multiprotein complex that triggers inflammatory signaling. NLRP3, in turn, is known to activate its downstream partner SGT1. In the current study, TRIM28 knockdown reduced the expression of both NLRP3 and SGT1, while overexpression raised their levels. Crucially, the researchers also examined functional readouts of inflammasome activation. TRIM28 knockdown decreased levels of cleaved caspase-1 and mature IL-1β, both hallmarks of inflammasome activity, and reduced secretion of IL-1β into the culture medium. Overexpression produced the opposite pattern. Co-immunoprecipitation experiments further revealed that TRIM28 physically interacts with NLRP3 and that TRIM28 overexpression significantly increased the SUMOylation of NLRP3, a modification previously linked to inflammasome stabilization.

With the macrophage-side mechanism established, the investigators turned to the keratinocyte side of the equation. They placed modified macrophages in the upper chamber of a Transwell system, separated by a porous membrane from HaCaT keratinocytes below, allowing soluble factors to pass but preventing direct cell contact. After 24 hours of co-culture, they measured keratinocyte viability and invasive capacity. The differences were dramatic. Co-culture with TRIM28-silenced macrophages reduced HaCaT cell viability to 60 percent of control levels, while co-culture with TRIM28-overexpressing macrophages raised viability to 140 percent. Invasion assays showed a similar pattern: silencing TRIM28 in macrophages reduced keratinocyte invasion to 60 percent of baseline, whereas overexpression increased it to 180 percent. These cellular behaviors, hyperproliferation and invasion, are relevant to inflammatory skin responses, although the authors are careful to note that they do not by themselves demonstrate a role for TRIM28 in psoriasis in living organisms.

To confirm that the NLRP3/SGT1 axis and SUMOylation were genuinely required for these effects rather than incidental, the researchers performed a series of rescue experiments. They knocked down NLRP3 or SGT1 in macrophages overexpressing TRIM28, or treated the cells with 2-D08, a chemical inhibitor of SUMOylation. Each intervention partially reversed the TRIM28-driven shift toward the pro-inflammatory macrophage phenotype, restoring the balance of CD86-positive and CD206-positive populations. The interventions also normalized cytokine secretion patterns, reduced cleaved caspase-1 and mature IL-1β levels, and attenuated the enhanced NLRP3 SUMOylation. Most importantly, these same interventions partially reversed the proliferative and invasive changes observed in co-cultured keratinocytes, confirming that the NLRP3/SGT1 pathway and SUMOylation are functionally required for the macrophage-mediated effects on skin cells.

The study places TRIM28 within a growing family of TRIM proteins implicated in inflammatory skin biology. Previous work has shown that TRIM14 promotes psoriasis-like inflammation by activating NF-κB signaling, and that TRIM27 amplifies IL-6/STAT3 signaling in keratinocytes. The current research extends this picture by suggesting that TRIM28 operates through a distinct mechanism, one involving post-translational modification of the NLRP3 inflammasome rather than transcriptional regulation alone. This positions TRIM28 as a potential bridging molecule between the epigenetic machinery of the cell and the innate immune response, a role that has parallels in other inflammatory conditions. The researchers draw comparisons to the HSP90β-SGT1 complex, which stabilizes NLRP3 in autoinflammatory syndromes, and to studies in fatty liver disease where HSP90 inhibition suppresses NLRP3 activation.

Nevertheless, the authors are emphatic about the limitations of their work. All experiments were performed in simplified in vitro systems using immortalized cell lines, which cannot fully replicate the cellular diversity, tissue architecture, and immune microenvironment of human psoriatic skin. The team did not examine TRIM28 expression in actual psoriatic lesions, did not analyze patient-derived samples, and did not correlate TRIM28 levels with disease severity. Macrophage polarization was assessed using a limited marker panel, and the authors acknowledge that the M1/M2 framework is a simplified operational model rather than a definitive classification of macrophage states. More comprehensive approaches, including single-cell RNA sequencing and multiplex flow cytometry, would be needed to fully characterize the macrophage phenotypes regulated by TRIM28. Additionally, the specific enzyme responsible for NLRP3 SUMOylation in this context remains unknown, as TRIM28 itself lacks SUMO ligase activity, suggesting intermediary proteins may be involved.

Looking forward, the researchers outline several priorities for future investigation. They propose examining TRIM28 expression and localization in human psoriatic tissue alongside clinical markers, validating the TRIM28–NLRP3/SGT1 axis in patient-derived cells and three-dimensional organotypic skin cultures, and testing whether modulating TRIM28 alters inflammation in animal models of psoriasis-like dermatitis. They also call for a more precise mapping of the molecular interactions between TRIM28, NLRP3, and SGT1, including identification of the relevant SUMO E3 ligase and assessment of downstream inflammasome assembly events. Until such work is completed, the current study stands as a rigorous piece of mechanistic cell biology, defining a TRIM28-dependent inflammatory pathway in a controlled laboratory setting. Its contribution is foundational rather than translational: it illuminates how a single regulatory protein can reshape the inflammatory conversation between two cell types central to skin immunity, while leaving open the critical question of whether that conversation drives human disease.

Subject of Research: TRIM28 regulation of macrophage polarization and keratinocyte behavior via the NLRP3/SGT1 axis in vitro

Article Title: TRIM28 Promotes Keratinocyte Proliferation and Invasion by Activating NLRP3/SGT1 Axis‐Mediated Macrophage Pro‐Inflammatory Polarization in an In Vitro Macrophage–Keratinocyte Co‐Culture Model

Article References: Gao, Z., Zhang, X., Wang, J., & Yang, N. (2026). TRIM28 Promotes Keratinocyte Proliferation and Invasion by Activating NLRP3/SGT1 Axis‐Mediated Macrophage Pro‐Inflammatory Polarization in an In Vitro Macrophage–Keratinocyte Co‐Culture Model. Immunity, Inflammation and Disease, 14(9), Article e70522. https://doi.org/10.1002/iid3.70522

Image Credits: AI Generated

DOI: 10.1002/iid3.70522

Keywords: TRIM28, macrophage polarization, NLRP3 inflammasome, SGT1, SUMOylation, keratinocytes, psoriasis, inflammation, IL-1β, co-culture, innate immunity, skin biology