Mice (Mus musculus) used in biomedical research are commonly housed at 19°C-23°C, below their thermoneutral zone (29°C-34°C), where metabolic homeostasis occurs. Although traditional housing exposes mice to chronic cold stress and modulates their immune response, the cellular mechanisms by which thermoneutrality shapes immune responses remain underdefined. CD4+ T cells are major contributors to host immunity through T cell receptor (TCR) signaling activated via recognition of peptide-major histocompatibility complex class II (pMHCII). We demonstrate that thermoneutral housing, compared to traditional housing, enhances tonic TCR signaling, upregulates genes associated with endogenous TCR stimulation, and increases TCR-driven TNF expression in CD4+ T cells. Mechanistically, these effects are in part dependent on tonic TCR engagement with self-pMHCII. In inflammatory disease models, thermoneutrality-driven increased CD4+ T cell TNF production correlates with amplified tissue inflammation. Together, these findings reveal how housing temperature may shape inflammatory responses through self-pMHCII-dependent TCR signaling in CD4+ T cells.

Uncontrolled inflammatory cytokine production promotes pathogenesis of various chronic diseases. Zinc (Zn) regulates immune cell inflammatory cytokine production. However, the influence of Zn on the inflammatory properties of non-immune cells known to contribute to disease pathogenesis is not well understood. Adipocytes respond to various immunological stimuli by activating inflammatory pathways and secreting inflammatory cytokines. Here, we investigated the impact of Zn on adipocyte inflammatory vigor. We show that treatment of primary mouse adipocytes with Zn, in the form of Zn pyrithione, restricted their toll-like receptor ligand-driven IL-6 production. Mechanistically, IL-6 secreted from adipocytes functions in an autocrine fashion to activate the Stat3 pathway and amplify IL-6 production via a positive feedback loop. Notably, Zn treatment of adipocytes suppressed Stat3 signaling activation to break the positive feedback loop and subsequent expression of IL-6 and its receptor genes (Il6st, Il6ra). Collectively, our findings uncover a novel inhibitory role for Zn in non-immune cell, specifically adipocyte, IL-6 production. These findings invoke a potential role of Zn in the regulation of adipocyte-associated chronic inflammation and disease pathogenesis.

BACKGROUND:Obesity-associated inflammation in white adipose tissue (WAT) worsens outcomes of influenza A virus (IAV) infection. A recently identified thoracic cavity WAT (tcWAT) supports IAV replication. However, tcWAT's immune cell composition, functional properties and role in IAV disease severity remains unclear. METHODS:Using a mouse model of diet-induced obesity, flow cytometry and single cell RNA-sequencing, we compared tcWAT with lung-distal visceral WAT assessing immune cell composition, transcriptomic profiles, inflammatory potential, and impact on IAV pathogenesis. RESULTS:At baseline, tcWAT was uniquely enriched for immune cells with heightened proinflammatory capacity and exhibited a striking predominance of lymphocytic populations, including immature and Satb1+ T cells, the latter expressing gene signatures associated with elevated T cell activation. Transfer of tcWAT immune cells into IAV infected recipients accelerated IAV disease severity. IAV infection robustly reshaped tcWAT immune landscape, driving expansion of a B cell population expressing Zbtb32 and upregulating genes involved in immune regulation and antiviral responses. Concurrently, IAV infection reduced immune populations linked to neutrophil regulation in tcWAT, while these same populations were expanded in the lung. CONCLUSIONS:These findings identify lung-proximal tcWAT as a distinct inflammatory tissue that may amplify pathogenic immune responses during IAV infection.

Processes that promote white adipocyte inflammatory function remain incompletely defined. Here, we demonstrated that type I interferon-dependent (IFN-I-dependent) skewing of adipocyte glycolysis, nicotinamide adenine dinucleotide (NAD+) utilization, and pyruvate kinase isozyme M2 (PKM2) function may contribute to increased systemic and tissue inflammation and disease severity in obesity. Notably, chemical and/or genetic inhibition of glycolysis, the NAD+ salvage pathway, or PKM2 restricted IFN-I-dependent increase in adipocyte inflammatory cytokine production. Further, genetic or small molecule targeting of PKM2 function in vivo was sufficient to reduce systemic and tissue inflammation and metabolic disease severity in obese mice, in an adipocyte PKM2-dependent manner. Further, white adipose tissue of individuals living with obesity and metabolic disease, compared with metabolically healthy individuals with obesity, showed an increase in expression of inflammatory and metabolic genes, while small molecule targeting of PKM2 function contributed to reduced IFN-I-driven inflammatory cytokine production by primary human adipocytes. Together, our findings invoke the IFN-I/PKM2 axis as a potential target for modulating adipocyte dysregulated inflammation.

BACKGROUND & AIM:To investigate the yet unexplored links between maternal and offspring outcomes of pregnancy metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS:Prospective observational study using magnetic resonance imaging with proton density fat fraction (MRI-PDFF) to determine the presence of maternal and offspring liver steatosis during gestation (3rd trimester) and in the first year postpartum. Women were deemed at high or low risk for MASLD if they had risk factors (pre-pregnancy obesity or MASLD, gestational diabetes, rapid weight gain during pregnancy). Multivariable regression and longitudinal mixed models assessed associations and changes in MRI-derived fat measures. RESULTS:We recruited 57 pregnant women; 47 (82 %) were high-risk for MASLD. MASLD was seen in 18 % (n = 10) of women (and 15 % [n = 7/47] of those high-risk), with a median PDFF of 5.9 % (IQR: 5.6-12.0 %). Maternal weight, BMI and HDL-C differed between groups. The fetal liver and placental PDFF of offspring of women with and without MASLD were not different. N = 7 (12 %) infants had clinically significant steatosis in utero, all offspring of mothers without gestational MASLD. Of the 30 mothers with repeat liver MRI-PDFF at 9 months postpartum, ten (33 %)had MASLD. N = 44 infants followed up at either 3 or 9 months of age, with only one still demonstrating significant steatosis 9 months. No infant exposed to gestational MASLD had significant steatosis in the first year. CONCLUSIONS:Gestational MASLD is generally mild, but hepatic fat fraction increases postpartum. Clinically significant hepatic steatosis occurs as early as in utero but is not associated with maternal MASLD.

Obesity is an independent risk factor for increased disease severity during influenza A virus (IAV) infection. White adipose tissue (WAT) inflammation promotes disease pathogenesis in obesity. Whether obesity modifies lung and WAT immune cells to amplify influenza severity is unknown. We show that obesity establishes a proinflammatory transcriptome in lung immune cells that is augmented during IAV infection and that IAV infection changes WAT immune cell milieu in obesity. Notably, a decrease in WAT macrophages (ATM) inversely correlates with an increase in infiltrating lung macrophages in obese IAV-infected mice. Further analyses of lung immune cell uncovered a macrophage subset that shares a transcriptomic signature with inflammatory ATMs. Importantly, adoptive transfer of ATMs from obese mice into lean IAV infected mice promotes host immune cell infiltration to the lungs. These findings suggest that, in an obese state, ATMs may exacerbate the inflammatory milieu important in pathologic responses to IAV infection.

Clinical evidence points to a function for B cell-activating factor (BAFF) in pregnancy. However, direct roles for BAFF-axis members in pregnancy have not been examined. Here, via utility of genetically modified mice, we report that BAFF promotes inflammatory responsiveness and increases susceptibility to inflammation-induced preterm birth (PTB). In contrast, we show that the closely related A proliferation-inducing ligand (APRIL) decreases inflammatory responsiveness and susceptibility to PTB. Known BAFF-axis receptors serve a redundant function in signaling BAFF/APRIL presence in pregnancy. Treatment with anti-BAFF/APRIL monoclonal antibodies or BAFF/APRIL recombinant proteins is sufficient to manipulate susceptibility to PTB. Notably, macrophages at the maternal-fetal interface produce BAFF, while BAFF and APRIL presence divergently shape macrophage gene expression and inflammatory function. Overall, our findings demonstrate that BAFF and APRIL play divergent inflammatory roles in pregnancy and provide therapeutic targets for mitigating risk of inflammation-induced PTB.

Introduction Inflammation is a common unifying factor in experimental models of non-alcoholic fatty liver disease (NAFLD) progression. Recent evidence suggests that housing temperature-driven alterations in hepatic inflammation correlate with exacerbated hepatic steatosis, development of hepatic fibrosis, and hepatocellular damage in a model of high fat diet-driven NAFLD. However, the congruency of these findings across other, frequently employed, experimental mouse models of NAFLD has not been studied. Methods Here, we examine the impact of housing temperature on steatosis, hepatocellular damage, hepatic inflammation, and fibrosis in NASH diet, methionine and choline deficient diet, and western diet + carbon tetrachloride experimental models of NAFLD in C57BL/6 mice. Results We show that differences relevant to NAFLD pathology uncovered by thermoneutral housing include: (i) augmented NASH diet-driven hepatic immune cell accrual, exacerbated serum alanine transaminase levels and increased liver tissue damage as determined by NAFLD activity score; (ii) augmented methionine choline deficient diet-driven hepatic immune cell accrual and increased liver tissue damage as indicated by amplified hepatocellular ballooning, lobular inflammation, fibrosis and overall NAFLD activity score; and (iii) dampened western diet + carbon tetrachloride driven hepatic immune cell accrual and serum alanine aminotransferase levels but similar NAFLD activity score. Discussion Collectively, our findings demonstrate that thermoneutral housing has broad but divergent effects on hepatic immune cell inflammation and hepatocellular damage across existing experimental NAFLD models in mice. These insights may serve as a foundation for future mechanistic interrogations focused on immune cell function in shaping NAFLD progression.

Influenza virus-induced respiratory pneumonia remains a major public health concern. Obesity, metabolic diseases, and female sex are viewed as independent risk factors for worsened influenza virus-induced lung disease severity. However, lack of experimental models of severe obesity in female mice limits discovery-based studies. Here, via utility of thermoneutral housing (30 degrees C) and highfat diet (HFD) feeding, we induced severe obesity and metabolic disease in female C57BL/6 mice and compared their responses to severely obese male C57BL/6 counterparts during influenza virus infection. We show that lean male and female mice have similar lung edema, inflammation, and immune cell infiltration during influenza virus infection. At standard housing conditions, HFD-fed male, but not female, mice exhibit severe obesity, metabolic disease, and exacerbated influenza disease severity. However, combining thermoneutral housing and HFD feeding in female mice induces severe obesity and metabolic disease, which is sufficient to amplify influenza virus-driven disease severity to a level comparable to severely obese male counterparts. Lastly, increased total body weights of male and female mice at time of infection correlated with worsened influenza virus-driven disease severity metrics. Together, our findings confirm the impact of obesity and metabolic disease as key risk factors to influenza disease severity and present a novel mouse experimental model suitable for future mechanistic interrogation of sex, obesity, and metabolic disease traits in influenza virus-driven disease severity.

Vertical transmission of obesity is a critical contributor to the unabated obesity pandemic and the associated surge in metabolic diseases. Existing experimental models insufficiently recapitulate “human-like” obesity phenotypes, limiting the discovery of how severe obesity in pregnancy instructs vertical transmission of obesity. Here, via utility of thermoneutral housing and obesogenic diet feeding coupled to syngeneic mating of WT obese female and lean male mice on a C57BL/6 background, we present a tractable, more “human-like” approach to specifically investigate how maternal obesity contributes to offspring health. Using this model, we found that maternal obesity decreased neonatal survival, increased offspring adiposity, and accelerated offspring predisposition to obesity and metabolic disease. We also show that severe maternal obesity was sufficient to skew offspring microbiome and create a proinflammatory gestational environment that correlated with inflammatory changes in the offspring in utero and adulthood. Analysis of a human birth cohort study of mothers with and without obesity and their infants was consistent with mouse study findings of maternal inflammation and offspring weight gain propensity. Together, our results show that dietary induction of obesity in female mice coupled to thermoneutral housing can be used for future mechanistic interrogations of obesity and metabolic disease in pregnancy and vertical transmission of pathogenic traits.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) exerts pleiotropic effects on macrophages and is required for self-renewal but the mechanisms responsible are unknown. Using GM-CSF receptor-β-chain deficient ( Csf2rb −/− ) mice, we show GM-CSF is critical for mitochondrial turnover, functions, and integrity. GM-CSF signaling is essential for fatty acid β-oxidation and markedly increased tricarboxylic acid cycle activity, oxidative phosphorylation, and ATP production. GM-CSF also regulated cytosolic pathways including glycolysis, pentose phosphate pathway, and amino acid synthesis. We conclude that GM-CSF regulates macrophages in part through a critical role in maintaining mitochondria, which are necessary for cellular metabolism as well as proliferation and self-renewal.

Objective The risks of excess sugar intake in addition to high-fat diet consumption on immunopathogenesis of obesity-associated metabolic diseases are poorly defined. Interleukin-4 (IL-4) and IL-13 signaling via IL-4Rα regulates adipose tissue lipolysis, insulin sensitivity, and liver fibrosis in obesity. However, the contribution of IL-4Rα to sugar rich diet-driven obesity and metabolic sequelae remains unknown. Methods WT, IL-4Rα-deficient (IL-4Rα −/− ) and STAT6-deficient mice (STAT6 −/− ) male mice were fed low-fat chow, high fat (HF) or HF plus high carbohydrate (HC/fructose) diet (HF + HC). Analysis included quantification of: (i) body weight, adiposity, energy expenditure, fructose metabolism, fatty acid oxidation/synthesis, glucose dysmetabolism and hepatocellular damage; (ii) the contribution of the hematopoietic or non-hematopoietic IL-4Rα expression; and (iii) the relevance of IL-4Rα downstream canonical STAT6 signaling pathway in this setting. Results We show that IL-4Rα regulated HF + HC diet-driven weight gain, whole body adiposity, adipose tissue inflammatory gene expression, energy expenditure, locomotor activity, glucose metabolism, hepatic steatosis, hepatic inflammatory gene expression and hepatocellular damage. These effects were potentially, and in part, dependent on non-hematopoietic IL-4Rα expression but were independent of direct STAT6 activation. Mechanistically, hepatic ketohexokinase-A and C expression was dependent on IL-4Rα, as it was reduced in IL-4Rα-deficient mice. KHK activity was also affected by HF + HC dietary challenge. Further, reduced expression/activity of KHK in IL-4Rα mice had a significant effect on fatty acid oxidation and fatty acid synthesis pathways. Conclusion Our findings highlight potential contribution of non-hematopoietic IL-4Rα activation of a non-canonical signaling pathway that regulates the HF + HC diet-driven induction of obesity and severity of obesity-associated sequelae.

Emerging evidence suggests a key contribution to non-alcoholic fatty liver disease (NAFLD) pathogenesis by Th17 cells. The pathogenic characteristics and mechanisms of hepatic Th17 cells, however, remain unknown. Here, we uncover and characterize a distinct population of inflammatory hepatic CXCR3+Th17 (ihTh17) cells sufficient to exacerbate NAFLD pathogenesis. Hepatic ihTh17 cell accrual was dependent on the liver microenvironment and CXCR3 axis activation. Mechanistically, the pathogenic potential of ihTh17 cells correlated with increased chromatin accessibility, glycolytic output, and concomitant production of IL-17A, IFNγ, and TNFα. Modulation of glycolysis using 2-DG or cell-specific PKM2 deletion was sufficient to reverse ihTh17-centric inflammatory vigor and NAFLD severity. Importantly, ihTh17 cell characteristics, CXCR3 axis activation, and hepatic expression of glycolytic genes were conserved in human NAFLD. Together, our data show that the steatotic liver microenvironment regulates Th17 cell accrual, metabolism, and competence toward an ihTh17 fate. Modulation of these pathways holds potential for development of novel therapeutic strategies for NAFLD.

Background Understanding gender-associated bias in aging and obesity-driven metabolic derangements has been hindered by the inability to model severe obesity in female mice. Methods Here, using chow- or high fat diet (HFD)-feeding regimens at standard (T S ) and thermoneutral (T N ) housing temperatures, the latter to model obesity in female mice, we examined the impact of gender and aging on obesity-associated metabolic derangements and immune responsiveness. Analysis included quantification of: (i) weight gain and adiposity; (ii) the development and severity of glucose dysmetabolism and non-alcoholic fatty liver disease (NAFLD); and (iii) induction of inflammatory pathways related to metabolic dysfunction. Results We show that under chow diet feeding regimen, aging was accompanied by increased body weight and white adipose tissue (WAT) expansion in a gender independent manner. HFD feeding regimen in aged, compared to young, male mice at T S , resulted in attenuated glucose dysmetabolism and hepatic steatosis. However, under T S housing conditions only aged, but not young, HFD fed female mice developed obesity. At T N however, both young and aged HFD fed female mice developed severe obesity. Independent of gender or housing conditions, aging attenuated the severity of metabolic derangements in HFD-fed obese mice. Tempered severity of metabolic derangements in aged mice was associated with increased splenic frequency of regulatory T (T reg ) cells, Type I regulatory (Tr1)-like cells and circulating IL-10 levels and decreased vigor of HFD-driven induction of inflammatory pathways in adipose and liver tissues. Conclusion Our findings suggest that aging-associated altered immunological profile and inflammatory vigor may play a dominant role in the attenuation of obesogenic diet-driven metabolic dysfunction.

The impact of immune mediators on weight homeostasis remains underdefined. Interrogation of resistance to diet-induced obesity in mice lacking a negative regulator of Toll-like receptor signaling serendipitously uncovered a role for B cell activating factor (BAFF). Here we show that overexpression of BAFF in multiple mouse models associates with protection from weight gain, approximating a log-linear dose response relation to BAFF concentrations. Gene expression analysis of BAFF-stimulated subcutaneous white adipocytes unveils upregulation of lipid metabolism pathways, with BAFF inducing white adipose tissue (WAT) lipolysis. Brown adipose tissue (BAT) from BAFF-overexpressing mice exhibits increased Ucp1 expression and BAFF promotes brown adipocyte respiration and in vivo energy expenditure. A proliferation-inducing ligand (APRIL), a BAFF homolog, similarly modulates WAT and BAT lipid handling. Genetic deletion of both BAFF and APRIL augments diet-induced obesity. Lastly, BAFF/APRIL effects are conserved in human adipocytes and higher BAFF/APRIL levels correlate with greater BMI decrease after bariatric surgery. Together, the BAFF/APRIL axis is a multifaceted immune regulator of weight gain and adipose tissue function.

White adipose tissue inflammation, in part via myeloid cell contribution, is central to obesity pathogenesis. Mechanisms regulating adipocyte inflammatory potential and consequent impact of such inflammation in disease pathogenesis remain poorly defined. We show that activation of the type I interferon (IFN)/IFNα receptor (IFNAR) axis amplifies adipocyte inflammatory vigor and uncovers dormant gene expression patterns resembling inflammatory myeloid cells. IFNβ-sensing promotes adipocyte glycolysis, while glycolysis inhibition impeded IFNβ-driven intra-adipocyte inflammation. Obesity-driven induction of the type I IFN axis and activation of adipocyte IFNAR signaling contributes to obesity-associated pathogenesis in mice. Notably, IFNβ effects are conserved in human adipocytes and detection of the type I IFN/IFNAR axis-associated signatures positively correlates with obesity-driven metabolic derangements in humans. Collectively, our findings reveal a capacity for the type I IFN/IFNAR axis to regulate unifying inflammatory features in both myeloid cells and adipocytes and hint at an underappreciated contribution of adipocyte inflammation in disease pathogenesis.

Infection-driven inflammation in pregnancy is a major cause of spontaneous preterm birth (PTB). Both systemic infection and bacterial ascension through the vagina/cervix to the amniotic cavity are strongly associated with PTB. However, the contribution of maternal or fetal inflammatory responses in the context of systemic or localized models of infection-driven PTB is not well defined. Here, using intraperitoneal or intraamniotic LPS challenge, we examined the necessity and sufficiency of maternal and fetal Toll-like receptor (TLR) 4 signaling in induction of inflammatory vigor and PTB. Both systemic and local LPS challenge promoted induction of inflammatory pathways in uteroplacental tissues and induced PTB. Restriction of TLR4 expression to the maternal compartment was sufficient for induction of LPS-driven PTB in either systemic or intraamniotic challenge models. In contrast, restriction of TLR4 expression to the fetal compartment failed to induce LPS-driven PTB. Vav1-Cre-mediated genetic deletion of TLR4 suggested a critical role for maternal immune cells in inflammation-driven PTB. Further, passive transfer of WT in vitro-derived macrophages and dendritic cells to TLR4-null gravid females was sufficient to induce an inflammatory response and drive PTB. Cumulatively, these findings highlight the critical role for maternal regulation of inflammatory cues in induction of inflammation-driven parturition.

Nonalcoholic fatty liver disease (NAFLD) is a major global health issue. Lack of effective therapies to NAFLD is attributed to limited knowledge of disease etiopathogenesis. Emerging evidence reveal key contribution of the Th17 axis in NAFLD. However, the characteristics and mechanisms underlying the pathogenic potential of Th17 cells in NAFLD remain unknown. Here, we uncover a unique population of inflammatory hepatic CXCR3+Th17 cells (ihTh17) that contribute to NAFLD progression. Longitudinal hepatic accrual of ihTh17 cells correlated with NAFLD severity in mice and was dependent on obesity and CXCR3 axis activation. ihTh17 cells, compared to conventional CXCR3-Th17 cells, exhibited increased open chromatin marks in enhancer regions of inflammatory and metabolic genes, increased glycolytic skewing and metabolic capacity, and were able to simultaneously produce an array of pro inflammatory cytokines that directly promoted hepatocellular damage. Adoptive transfer of ihTh17 cells accelerated and exacerbated NAFLD pathogenesis in vivo. Modulation of the metabolic state of ihTh17 cells was sufficient to reverse ihTh17-centric inflammatory vigor and NAFLD severity. Importantly, ihTh17 cell hepatic accrual and inflammatory capacity, CXCR3 axis activation, and hepatic expression of glycolysis-associated genes were conserved in humans with NAFLD and positively correlated with disease severity. In sum, we show that the obesity-associated environment regulates Th17 cell metabolism and competence towards a unique inflammatory hepatic Th17 fate in turn contributing to NAFLD progression. Notably, modulation of these pathways holds potential for development of novel therapeutic strategies to NAFLD. Disclosure M.E. Moreno-Fernandez: None. J. Oates: None. M.S. Damen: None. J.R. Doll: None. T.E. Stankiewicz: None. S. Divanovic: Consultant; Self; Janssen Pharmaceuticals, Inc., Pfizer Inc. Funding American Diabetes Association (1-19-PMF-019 to M.E.M-F.); National Institutes of Health (R01DK099222, R01DK099222-02S1P30, DK078392)