FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation.

Fatty acid binding protein 4 (FABP4), a member of a family of lipid-binding proteins, is known to play a role in inflammation by virtue of its ability to regulate intracellular events such as lipid fluxes and signaling. Studies have indicated a proinflammatory role for FABP4 in allergic asthma although its expression and function in eosinophils, the predominant inflammatory cells recruited to allergic airways, were not investigated. We examined expression of FABP4 in murine eosinophils and its role in regulating cell recruitment in vitro as well as in cockroach antigen (CRA)-induced allergic airway inflammation. CRA exposure led to airway recruitment of FABP4-expressing inflammatory cells, specifically eosinophils, in wild-type (WT) mice. FABP4 expression in eosinophils was induced by TNF-α as well as IL-4 and IL-13. FABP4-deficient eosinophils exhibited markedly decreased cell spreading/formation of leading edges on vascular cell adhesion molecule-1 and significantly decreased adhesion to intercellular adhesion molecule-1 associated with reduced ß2-integrin expression relative to WT cells. Furthermore, FABP4-deficient eosinophils exhibited decreased migration, F-actin polymerization, calcium flux, and ERK(1/2) phosphorylation in response to eotaxin-1. In vivo, CRA-challenged FABP4-deficient mice exhibited attenuated eosinophilia and significantly reduced airway inflammation (improved airway reactivity, lower IL-5, IL-13, TNF-α, and cysteinyl leukotriene C4 levels, decreased airway structural changes) compared with WT mice. In conclusion, expression of FABP4 in eosinophils is induced during conditions of inflammation and plays a proinflammatory role in the development of allergic asthma by promoting eosinophil adhesion and migration and contributing to the development of various aspects of airway inflammation.

A Research Operations, Management, and Strategy Fellowship for Life Sciences PhD Graduates.

PROBLEM: With less than 25% of PhD-trained scientists in the United States securing a tenure-track faculty position following training, nonacademic careers have become common. As the academic research enterprise has increased, business-oriented careers have emerged. The Research Operations, Management, and Strategy (ROMS) Fellowship was developed to increase awareness of and prepare life sciences PhD graduates for business-focused careers. APPROACH: The ROMS Fellowship was developed from March through December 2018 by the University of Michigan Medical School. Launched in 2019 and based on real-world experiences, the 2-year ROMS Fellowship combines immersion rotations and project work to develop an understanding of foundational infrastructure across the full spectrum of research. OUTCOMES: From 2019 to 2022, there were 4 ROMS Fellowship recruitment cycles, with a mean of 7 applicants per cycle and 2 fellows selected each year. Of the 8 fellows recruited, 5 (62.5%) joined directly from PhD training, whereas 3 (37.5%) had 2 to 6 years of postdoctoral training. Fellows have worked with 26 departments on 44 rotation projects and 30 impact projects and self-reported significant skill development in communicating with diverse stakeholders, strategic thinking, using new tools and resources, developing and scoping a project plan, and managing and leading a project. To date, 4 fellows have completed the program and were hired immediately into full-time positions at the University of Michigan Medical School. NEXT STEPS: Early feedback indicates that the program has been well received and effective. Previously, program refinement was directed by qualitative input from fellows and unit directors. However, for future cohorts, assessment tools will be implemented to capture qualitative and quantitative data to measure acquired skills and how program components contribute to professional development and career placement. A longitudinal follow-up will also be conducted with program alumni to track longer-term outcomes and career pathways.

Mobilizing cross-disciplinary teams to advance translational research using design thinking methods.

Funding agencies are increasingly seeking team-based approaches to tackling complex research questions, but there is a need to mobilize translational teams and create shared visions and strategic action plans long before specific funding opportunities are considered or even released. This is particularly evident for teams who want to pursue large-scale grants, where cross-disciplinary synergy is often required. In response, we created Research Jams, which are engaging yet structured brainstorming sessions that bring together groups for the first time to collectively generate novel research ideas, critically map the future of initiatives, prioritize opportunities and next steps, and build community. Research Jams leveraged various aspects of design thinking, including divergence and convergence, visual thinking, and amplifying diversity. We piloted seven Research Jams for a collective 129 researchers, staff, and partners across 50 University of Michigan units and external organizations. Feedback was overwhelmingly positive, with the vast majority of survey respondents indicating that the sessions were helpful for surfacing shared ideas or visions and that opportunities emerged they would like to pursue. Research Jams were ideal for cross-disciplinary groups who wanted to collaboratively ideate and strategize around complex problems in translational research. Importantly, these models have the potential for implementation with groups in any disciplinary domain who want to spur collaborations to address challenging problems. Our ultimate goal is for Research Jams to be the first intervention within a comprehensive support pathway that extends from early brainstorming all the way to grant submission.

A structured professional development curriculum for postdoctoral fellows leads to recognized knowledge growth.

Postdoctoral training enables research independence and professional readiness. National reports have emphasized professional development as a critical component of this training period. In response, many institutions are establishing transferable skills training workshops for postdocs; however, the lack of structured programs and an absence of methods to assess outcomes beyond participant satisfaction surveys are critical gaps in postdoctoral training. To address these shortcomings, we took the approach of structured programming and developed a method for controlled assessment of outcomes. Our program You3 (You, Your Team, Your Project), co-designed by postdoctoral fellows, focused on discussing specific management and leadership skills agnostic of ultimate career path(s) in a structured manner. We then measured outcomes in a controlled manner, by systematically comparing perceived knowledge and growth as indicators of awareness and confidence in participants against that of non-participants as the control group. You3 participants self-rated greater growth in targeted competencies compared to non-participants independent of the number of years of training. This growth was shown by multiple criteria including self-reporting and associative analysis. Correspondingly, You3 participants reported greater knowledge in 75% of the modules when compared to controls. These data indicate that structured learning, where postdocs commit to a curriculum via a cohort-structure, leads to positive outcomes and provides a framework for programs to assess outcomes in a rigorous manner.

A role for keratins in supporting mitochondrial organization and function in skin keratinocytes.

Mitochondria fulfill essential roles in ATP production, metabolic regulation, calcium signaling, generation of reactive oxygen species (ROS), and additional determinants of cellular health. Recent studies have highlighted a role for mitochondria during cell differentiation, including in skin epidermis. The observation of oxidative stress in keratinocytes from Krt16 null mouse skin, a model for pachyonychia congenita (PC)-associated palmoplantar keratoderma, prompted us to examine the role of Keratin (K) 16 protein and its partner K6 in regulating the structure and function of mitochondria. Electron microscopy revealed major anomalies in mitochondrial ultrastructure in late stage, E18.5, Krt6a/Krt6b null embryonic mouse skin. Follow-up studies utilizing biochemical, metabolic, and live imaging readouts showed that, relative to controls, skin keratinocytes null for Krt6a/Krt6b or Krt16 exhibit elevated ROS, reduced mitochondrial respiration, intracellular distribution differences, and altered movement of mitochondria within the cell. These findings highlight a novel role for K6 and K16 in regulating mitochondrial morphology, dynamics, and function and shed new light on the causes of oxidative stress observed in PC and related keratin-based skin disorders.

Non-canonical processes that shape the cell migration landscape.

Migration is a vital, intricate, and multi-faceted process that involves the entire cell, entails the integration of multiple external cues and, at times, necessitates high-level coordination among fields of cells that can be physically attached or not, depending on the physiological setting. Recent advances have highlighted the essential role of cellular components that have not been traditionally considered when studying cell migration. This review details how much we recently learned by studying the role of intermediate filaments, the nucleus, extracellular vesicles, and mitochondria during cell migration.

FABP4/aP2 Regulates Macrophage Redox Signaling and Inflammasome Activation via Control of UCP2.

Obesity-linked metabolic disease is mechanistically associated with the accumulation of proinflammatory macrophages in adipose tissue, leading to increased reactive oxygen species (ROS) production and chronic low-grade inflammation. Previous work has demonstrated that deletion of the adipocyte fatty acid-binding protein (FABP4/aP2) uncouples obesity from inflammation via upregulation of the uncoupling protein 2 (UCP2). Here, we demonstrate that ablation of FABP4/aP2 regulates systemic redox capacity and reduces cellular protein sulfhydryl oxidation and, in particular, oxidation of mitochondrial protein cysteine residues. Coincident with the loss of FABP4/aP2 is the upregulation of the antioxidants superoxide dismutase (SOD2), catalase, methionine sulfoxide reductase A, and the 20S proteasome subunits PSMB5 and αß. Reduced mitochondrial protein oxidation in FABP4/aP2-/- macrophages attenuates the mitochondrial unfolded-protein response (mtUPR) as measured by expression of heat shock protein 60, Clp protease, and Lon peptidase 1. Consistent with a diminished mtUPR, FABP4/aP2-/- macrophages exhibit reduced expression of cleaved caspase-1 and NLRP3. Secretion of interleukin 1ß (IL-1ß), in response to inflammasome activation, is ablated in FABP4/aP2-/- macrophages, as well as in FABP4/aP2 inhibitor-treated cells, but partially rescued in FABP4/aP2-null macrophages when UCP2 is silenced. Collectively, these data offer a novel pathway whereby FABP4/aP2 regulates macrophage redox signaling and inflammasome activation via control of UCP2 expression.

Loss of Fatty Acid Binding Protein 4/aP2 Reduces Macrophage Inflammation Through Activation of SIRT3.

Activation of proinflammatory macrophages plays an important role in the pathogenesis of insulin resistance, type 2 diabetes, and atherosclerosis. Previous work using high fat-fed mice has shown that ablation of the adipocyte fatty acid binding protein (FABP4/aP2) in macrophages leads to an antiinflammatory state both in situ and in vivo, and the mechanism is linked, in part, to increased intracellular monounsaturated fatty acids and the up-regulation of uncoupling protein 2. Here, we show that loss of FABP4/aP2 in macrophages additionally induces sirtuin 3 (SIRT3) expression and that monounsaturated fatty acids (C16:1, C18:1) lead to increased SIRT3 protein expression. Increased expression of SirT3 in FABP4/aP2 null macrophages occurs at the protein level with no change in SirT3 mRNA. When compared with controls, silencing of SIRT3 in Raw246.7 macrophages leads to increased expression of inflammatory cytokines, inducible nitric oxide synthase and cyclooxygenase 2. In contrast, loss of SIRT3 in FABP4/aP2-deficient macrophages attenuates the suppressed inflammatory signaling, reduced reactive oxygen species production, lipopolysaccharide-induced mitochondrial dysfunction, and increased fatty acid oxidation. These results suggest that the antiinflammatory phenotype of FABP4/aP2 null mice is mediated by increased intracellular monounsaturated fatty acids leading to the increased expression of both uncoupling protein 2 and SirT3.

Uncoupling lipid metabolism from inflammation through fatty acid binding protein-dependent expression of UCP2.

Chronic inflammation in obese adipose tissue is linked to endoplasmic reticulum (ER) stress and systemic insulin resistance. Targeted deletion of the murine fatty acid binding protein (FABP4/aP2) uncouples obesity from inflammation although the mechanism underlying this finding has remained enigmatic. Here, we show that inhibition or deletion of FABP4/aP2 in macrophages results in increased intracellular free fatty acids (FFAs) and elevated expression of uncoupling protein 2 (UCP2) without concomitant increases in UCP1 or UCP3. Silencing of UCP2 mRNA in FABP4/aP2-deficient macrophages negated the protective effect of FABP loss and increased ER stress in response to palmitate or lipopolysaccharide (LPS). Pharmacologic inhibition of FABP4/aP2 with the FABP inhibitor HTS01037 also upregulated UCP2 and reduced expression of BiP, CHOP, and XBP-1s. Expression of native FABP4/aP2 (but not the non-fatty acid binding mutant R126Q) into FABP4/aP2 null cells reduced UCP2 expression, suggesting that the FABP-FFA equilibrium controls UCP2 expression. FABP4/aP2-deficient macrophages are resistant to LPS-induced mitochondrial dysfunction and exhibit decreased mitochondrial protein carbonylation and UCP2-dependent reduction in intracellular reactive oxygen species. These data demonstrate that FABP4/aP2 directly regulates intracellular FFA levels and indirectly controls macrophage inflammation and ER stress by regulating the expression of UCP2.