Characterizing the Effects of Vaccine Adjuvants on Skeletal Muscle Myotubes and Macrophages

Vaccines train the immune system to recognize and defend against pathogens. Currently, six types of vaccines are in use and include live-attenuated, inactivated, viral vector, protein subunit, toxoid, and messenger RNA (mRNA), the latter of which was recently approved for humans during the COVID-19 pandemic. To increase the longevity and magnitude of immune responses, some vaccines are combined with adjuvants. Mouse models have shown that adjuvants in combination with antigens can elicit a pro-inflammatory immune system response that is required for proper development of protective immunity. There has been recent appreciation for the immunomodulatory functions of skeletal muscle, yet their contribution to the immunology of vaccination remains incompletely understood. Considering most vaccines are administered intramuscularly, we utilized C2C12 mouse myotubes and J774 macrophages to explore the cytokine response that skeletal muscle cells and macrophages evoke in response to several types of vaccine adjuvants in absence of the antigen. C2C12 myotubes and J774 macrophages were treated with 7 commonly used adjuvants or appropriate controls and collected at 6 hours and 24 hours. Cytokine secretion, cytotoxicity, and effects on myotube diameter were analyzed. Most adjuvants, except for the positive controls (LPS and PAM3CSK4), CpG 1826, and Quil-A, did not induce a pro-inflammatory response in C2C12 myotubes or J774 macrophages. Interestingly, LPS, PAM3CSK4, MF59, Quil-A, AS03, and CFA, led to increases in C2C12 myotube diameter indicating an activation of hypertrophy. The lack of pro-inflammatory effects indicates that most adjuvants need antigens or additional cell-cell interactions at the injection site to produce a pro-inflammatory cytokine response

The Endogenous Th17 Response in NO<inf>2</inf>-Promoted Allergic Airway Disease Is Dispensable for Airway Hyperresponsiveness and Distinct from Th17 Adoptive Transfer

Severe, glucocorticoid-resistant asthma comprises 5-7% of patients with asthma. IL-17 is a biomarker of severe asthma, and the adoptive transfer of Th17 cells in mice is sufficient to induce glucocorticoid-resistant allergic airway disease. Nitrogen dioxide (NO2) is an environmental toxin that correlates with asthma severity, exacerbation, and risk of adverse outcomes. Mice that are allergically sensitized to the antigen ovalbumin by exposure to NO2 exhibit a mixed Th2/Th17 adaptive immune response and eosinophil and neutrophil recruitment to the airway following antigen challenge, a phenotype reminiscent of severe clinical asthma. Because IL-1 receptor (IL-1R) signaling is critical in the generation of the Th17 response in vivo, we hypothesized that the IL-1R/Th17 axis contributes to pulmonary inflammation and airway hyperresponsiveness (AHR) in NO2-promoted allergic airway disease and manifests in glucocorticoid-resistant cytokine production. IL-17A neutralization at the time of antigen challenge or genetic deficiency in IL-1R resulted in decreased neutrophil recruitment to the airway following antigen challenge but did not protect against the development of AHR. Instead, IL-1R-/- mice developed exacerbated AHR compared to WT mice. Lung cells from NO2-allergically inflamed mice that were treated in vitro with dexamethasone (Dex) during antigen restimulation exhibited reduced Th17 cytokine production, whereas Th17 cytokine production by lung cells from recipient mice of in vitro Th17-polarized OTII T-cells was resistant to Dex. These results demonstrate that the IL-1R/Th17 axis does not contribute to AHR development in NO2-promoted allergic airway disease, that Th17 adoptive transfer does not necessarily reflect an endogenously-generated Th17 response, and that functions of Th17 responses are contingent on the experimental conditions in which they are generated. © 2013 Martin et al

Glutathione-S-transferase P promotes glycolysis in asthma in association with oxidation of pyruvate kinase M2

Background: Interleukin-1-dependent increases in glycolysis promote allergic airways disease in mice and disruption of pyruvate kinase M2 (PKM2) activity is critical herein. Glutathione-S-transferase P (GSTP) has been implicated in asthma pathogenesis and regulates the oxidation state of proteins via S-glutathionylation. We addressed whether GSTP-dependent S-glutathionylation promotes allergic airways disease by promoting glycolytic reprogramming and whether it involves the disruption of PKM2. Methods: We used house dust mite (HDM) or interleukin-1β in C57BL6/NJ WT or mice that lack GSTP. Airway basal cells were stimulated with interleukin-1β and the selective GSTP inhibitor, TLK199. GSTP and PKM2 were evaluated in sputum samples of asthmatics and healthy controls and incorporated analysis of the U-BIOPRED severe asthma cohort database. Results: Ablation of Gstp decreased total S-glutathionylation and attenuated HDM-induced allergic airways disease and interleukin-1β-mediated inflammation. Gstp deletion or inhibition by TLK199 decreased the interleukin-1β-stimulated secretion of pro-inflammatory mediators and lactate by epithelial cells. 13C-glucose metabolomics showed decreased glycolysis flux at the pyruvate kinase step in response to TLK199. GSTP and PKM2 levels were increased in BAL of HDM-exposed mice as well as in sputum of asthmatics compared to controls. Sputum proteomics and transcriptomics revealed strong correlations between GSTP, PKM2, and the glycolysis pathway in asthma. Conclusions: GSTP contributes to the pathogenesis of allergic airways disease in association with enhanced glycolysis and oxidative disruption of PKM2. Our findings also suggest a PKM2-GSTP-glycolysis signature in asthma that is associated with severe disease

Serum amyloid A3 is required for normal weight and immunometabolic function in mice.

Serum amyloid A (SAA) is an apolipoprotein that is robustly upregulated in numerous inflammatory diseases and has been implicated as a candidate pro-inflammatory mediator. However, studies comparing endogenous SAAs and recombinant forms of the acute phase protein have generated conflicting data on the function of SAA in immunity. We generated SAA3 knockout mice to evaluate the contribution of SAA3 to immune-mediated disease, and found that mice lacking SAA3 develop adult-onset obesity and metabolic dysfunction along with defects in innate immune development. Mice that lack SAA3 gain more weight, exhibit increased visceral adipose deposition, and develop hepatic steatosis compared to wild-type littermates. Leukocytes from the adipose tissue of SAA3-/- mice express a pro-inflammatory phenotype, and bone marrow derived dendritic cells from mice lacking SAA3 secrete increased levels of IL-1β, IL-6, IL-23, and TNFα in response to LPS compared to cells from wild-type mice. Finally, BMDC lacking SAA3 demonstrate an impaired endotoxin tolerance response and inhibited responses to retinoic acid. Our findings indicate that endogenous SAA3 modulates metabolic and immune homeostasis

Lack of SAA3 inhibits dendritic cell responses to retinoic acid.

<p>Bone marrow derived cells were isolated from WT and SAA3-/- mice and differentiated into dendritic cells for 7 days in the presence of 5% GM-CSF or 5% GM-CSF + 0.1 μM all-trans-retinoic acid (ATRA). At day 7, cells were analyzed for <i>Saa3</i>, <i>Rara</i>, and <i>Rarb</i> gene expression by Q-PCR (A). At day 7, cells were also challenged with 100 ng/ml LPS for 24 hours, and IL-10, IL-1β, and TNFα were measured from cell supernatants by ELISA (B). n = 3-7/group. * = p<0.05, ** = p<0.01, *** = p<0.005, **** = p<0.001.</p

High fat diet leads to more substantial weight gain and insulin-sensitive gene repression in SAA3-/- mice.

<p>18 week old wild type (WT) and SAA3-/- mice were placed on high-fat diet (HFD = 60% kCal from fat) or low-fat diet (LFD = 10% kCal from fat) for 7 days. Total body weight was measured each day (A). Comparisons displayed are between HFD-fed WT and SAA3-/- mice. The expression of SAA isoforms in VAT was analyzed from WT mice on the LFD and HFD (B). Insulin-sensitive gene expression was analyzed by Q-PCR from VAT, subcutaneous adipose tissue (SCAT), or liver of WT and SAA3-/- mice fed HFD (C). n = 4-5/group. * = p<0.05, ** = p<0.01, *** = p<0.005, **** = p<0.001.</p

SAA3-/- mice display altered myeloid cell responses.

<p>Bone marrow derived dendritic cells generated from WT and SAA3-/- mice were challenged with increasing doses of LPS for 4 and 24 hours. IL-1β, IL-6, IL-23, and TNFα were measured from cell supernatants by ELISA (A). BMDC were challenged for 24 hours with increasing doses of LPS (24h). These cells were then restimulated with the same dose of LPS for a further 24 hours (Tolerized) and supernatants were analyzed for IL-1β, IL-10, and TNFα by ELISA (B). n = 3-7/group. * = p<0.05, ** = p<0.01, *** = p<0.005, **** = p<0.001.</p

Characterization of SAA3-/- mice.

<p>Levels of basal <i>Saa3</i> expression from wild-type (WT) mice were measured from VAT, lung, liver, spleen, and kidney. Relative levels were compared to the kidney (A). WT and SAA3 knockout (SAA3-/-) littermates were analyzed by Q-PCR for <i>Saa3</i> gene expression in the same organs to confirm the deletion of <i>Saa3</i> in the SAA3-/- mice (B). Characterization of different SAA isoforms was performed by Q-PCR from VAT (C). WT and SAA3-/- mice were analyzed at 18 weeks of age for total body weight (D) and VAT weight (E). Insulin sensitive gene expression was analyzed by Q-PCR from VAT (F). Adipocyte and stromal vascular fractions were confirmed for cell-specific genes in wild-type mice (G). SVF from the VAT of WT and SAA3-/- mice were analyzed by Q-PCR for pro-inflammatory genes (H) and M2-related genes (I). Serum from WT and SAA3-/- mice was analyzed for circulating cytokines (J) n = 3-9/group. * = p<0.05, ** = p<0.01, *** = p<0.005, **** = p<0.001.</p

Livers from SAA3-/- mice demonstrate early signs of non-alcoholic fatty liver disease.

<p>Liver tissue from WT and SAA3-/- mice at 18 weeks of age were fixed in 10% neutral-buffered formalin and stained for H&E (A, top row) and Oil-Red O (A, bottom row). Representative images are presented. Q-PCR analysis from liver was performed for genes that regulate insulin sensitivity (B). n = 3-4/group. *p = <0.05, **** = p<0.001.</p