Role of CXCL7 in obesity and metabolic disease

Abstract

Obesity is a multifactorial disease marked by chronic low-grade inflammation and profound metabolic disturbances that heighten the risk for insulin resistance, cardiovascular disease, and type 2 diabetes. A growing body of literature has implicated immune-inflammatory responses as central mediators of obesity-associated metabolic dysfunction. However, the complexity of adipose tissue remodeling, immune cell infiltration, and chemokine signaling remains poorly defined. This dissertation encompasses two interconnected projects aimed at elucidating molecular mechanisms driving obesity-related inflammation and insulin resistance, with a focus on optimizing in vitro adipocyte models and dissecting the role of the platelet- derived chemokine CXCL7 in adipose tissue physiology.

Project One: Optimization of an In vitro Model of Inflammation and Insulin Resistance in Adipocytes The first phase of this work focused on developing a reproducible in vitro model of adipocyte inflammation using differentiated 3T3-L1 cells exposed to tumor necrosis factor-alpha (TNF-α) and hypoxia. Recognizing the limitations of prolonged cytokine exposure, we optimized the model by shortening TNF-α treatment duration to 12 hours-reducing cell death while preserving pro-inflammatory signaling and insulin resistance features. Glucose uptake assays, RT-qPCR, and ELISA demonstrated that the 12-hour TNF-α/hypoxia protocol sufficiently induced inflammatory gene expression and suppressed insulin-responsive markers such as Glut4 and Adipoq. Moreover, conditioned media collected from these cultures contained elevated levels of secreted inflammatory chemokines, validating the model's potential for future co- culture studies. This refined model provides a robust platform for investigating paracrine signaling between inflamed adipocytes and other cells type and sets the stage for mechanistic studies into adipose tissue crosstalk. Project Two: Investigating the Role of CXCL7 in Obesity and Metabolic Disease To interrogate the physiological role of CXCL7, we utilized genetically modified mice in a C57BL/6N background: wild-type (WT), CXCL7 knockout (KO), and KO mice rescued with a human CXCL7 transgene (KO+TG). Both male and female mice were fed either a standard chow diet or a Western diet (WD) for 12 weeks. Our comprehensive phenotyping approach included body weight tracking, metabolic cage assessments, glucose tolerance testing, tissue collection, and transcriptomic profiling of inguinal white adipose tissue (iWAT) and liver. In male KO mice, Western diet feeding led to pronounced increases in body weight, adiposity, and tissue mass across several depots, indicating heightened susceptibility to diet-induced obesity. In contrast, KO+TG males expressing human CXCL7 exhibited partial protection from these effects. Female mice also exhibited a CXCL7-dependent pattern, though the magnitude of difference between genotypes was less striking than in males. Linear regression modeling confirmed that iWAT was the most genotype-sensitive depot in both sexes, making it a sentinel issue for CXCL7-dependent regulation of adiposity. Histological analysis revealed hypertrophic adipocytes in WD-fed KO mice and a reversal of this phenotype in KO+TG animals. RNA sequencing of iWAT indicated that KO mice had suppressed immune gene expression and upregulated oxidative phosphorylation and mitochondrial function pathways, whereas CXCL7 expression in KO+TG mice reprogrammed the transcriptome toward inflammatory and immune-enriched signaling, including cytokine signaling, NF-κB activation, and MHC class II antigen presentation. These transcriptional changes were accompanied by increased immune cell infiltration, as confirmed by computational deconvolution of bulk RNA-seq data. Notably, neutrophils—known early responders in inflammation remained enriched in KO+TG iWAT under chronic WD exposure, suggesting prolonged CXCL7-dependent immune activation. Importantly, we also assessed the liver, a central metabolic organ heavily impacted by obesity. Liver transcriptomic analysis revealed that WD feeding induced strong inflammatory and metabolic remodeling in all genotypes, but CXCL7 expression uniquely amplified platelet activation and immune signaling pathways in KO+TG mice. KEGG enrichment analyses indicated that genes associated with platelet activation, cytokine-cytokine receptor interaction, and leukocyte recruitment were significantly upregulated in the livers of KO+TG_WD mice compared to both KO and WT counterparts. GSEA confirmed a marked enrichment of platelet activation pathways, implicating CXCL7 as a systemic regulator beyond adipose tissue. Furthermore, Western diet feeding consistently upregulated circulating CXCL7 and the platelet- derived form. The elevated expression of platelet-related genes and inflammatory chemokines supports a model in which CXCL7 serves as a key mediator linking platelet activation to chronic inflammation in obesity. Conclusions Together, these two projects provide compelling evidence that CXCL7 plays a role in adipose biology and systemic inflammation. Our in vitro model establishes a platform for studying adipocyte-inflammation and other cells interaction, while our in vivo studies demonstrate that CXCL7 promotes pro-inflammatory remodeling of adipose and hepatic tissues, especially under dietary stress. Notably, reconstitution of human CXCL7 in KO mice partially restored wild-type-like metabolic phenotypes while also revealing unique inflammatory responses distinct from the murine gene. This dissertation highlights CXCL7 as a diet-inducible chemokine that shapes adipose tissue remodeling, immune infiltration, and metabolic outcomes in a sex- and depot-specific manner. Future work will focus on dissecting the CXCL7-CXCR2 signaling axis and exploring its therapeutic potential in metabolic diseases marked by immune dysregulation, including obesity, insulin resistance, and metabolic dysfunction fatty liver disease (MAFLD).