T and B cells are markedly decreased in the VAT of CD11c-MyD88 KO DIO mice.

<p>(A-E) SVF obtained from the epididymal fat pads of Cre- and Cre+ mice fed a HFD for the indicated lengths of time were analyzed by flow cytometry to quantify the numbers of total T cells (A), B cells (B), CD4⁺ T cells (C), CD8⁺ T cells (D), and Tregs (E); the data are expressed as number of cells per gram of fat (wet weight). n = 5 mice per group (*, <i>p</i> < 0.05 <i>vs</i>. Cre- group; ns, no significant difference <i>vs</i>. Cre- group). (F-G) qRT-PCR analysis of mRNA expression of <i>Ifng</i>, <i>Il2</i>, <i>Il17</i>, <i>Ccl17</i>, <i>Ccl19</i>, and <i>Ccl22</i> in SVF obtained from epididymal fat pad of 10 wk HFD-fed Cre- and Cre+ mice. n = 5 mice per group (*, <i>p</i> < 0.05 <i>vs</i>. Cre- group; ns, no significant difference <i>vs</i>. Cre- group).</p

CD11c⁺ F4/80^- and CD11c⁺ F4/80⁺ cells from the VAT of lean and obese mice have characteristics of dendritic cells and macrophages, respectively.

<p>(A) Representative data of flow cytometric analysis of SVF cells obtained from the epididymal fat pad of 16 wk/o chow-fed male C57BL/6J mice. The dot-plot shows CD11c and F4/80 immunostaining in CD45⁺-gated SVF cells. (<i>B</i>) Representative data of flow-cytometric analysis of cell surface expression of Mertk, CD64, and CCR7 in CD11c⁺F4/80⁺ and CD11c⁺F4/80^- SVF cells. n = 3. (C and D) Representative data of flow cytometric analysis (contour plot) of cell-surface MHC-II and CD86 respectively in the indicated population of SVF cells. n = 3. (E) FACS-sorted splenic DCs (Sp DC) and VAT-SVF-derived CD11c⁺ F4/80⁺ and CD11c⁺ F4/80^- cells from lean mice were loaded with OVA and co-cultured with CFSE-labeled naïve CD4⁺ OTII transgenic T cells for 72 h. The histograms indicate proliferation of transgenic T cells as measured by dilution of CFSE dye. The bar graph represents the quantified data (*, p < 0.05 <i>vs</i>. CD11c⁺F4/80⁺ group; # p < 0.05 <i>vs</i>. Splenic DC-No Ova group). The data are representative of two independent experiments. (F) IFN-γ ELISA of cell culture supernatants of the cells used for the experiment in panel D. (G) FACS-sorted splenic DCs (Sp DC) and VAT-SVF-derived CD11c⁺ F4/80⁺ and CD11c⁺F4/80^- cells from DIO mice were loaded with OVA and co-cultured with CFSE-labeled naïve CD4⁺ OTII transgenic T cells for 72 h. Splenic DCs without ovalbumin served as control (*, p < 0.05 <i>vs</i>. CD11c⁺F4/80⁺ group; #, p < 0.05 <i>vs</i>. splenic DC- No Ova group). The data are representative of two independent experiments.</p

Absence of systemic immune and inflammatory changes in CD11c-MyD88 KO DIO mice.

<p>(A-C) Total splenocyte count, percent splenic macrophages (Mφ), DCs, T cells, B cells, and Tregs in Cre- and Cre+ DIO mice. n = 5 mice per group (ns, no significant difference <i>vs</i>. Cre- group). (D-E) qRT-PCR analysis of <i>Tnfa</i>, <i>Il6</i>, <i>Mcp1</i>, <i>Il10</i>, <i>and Tgfb</i> mRNA in the spleens and livers of Cre- and Cre+ DIO mice. n = 3 mice per group (ns, no significant difference <i>vs</i>. Cre- group). (F) ELISA-based measurement of TNF, IL-6, MCP-1, IL-1β, and IL-10 in serum of Cre- and Cre+ DIO mice. n = 5 mice per group (ns, no significant difference <i>vs</i>. Cre- group).</p

Macrophages and dendritic cell numbers and myeloid cell-derived cytokines are not altered in the VAT of CD11c-MyD88 KO DIO mice.

<p>(A-B) SVF obtained from the epididymal fat pads of Cre- and Cre+ mice fed a HFD for the indicated lengths of time were analyzed by flow cytometry to quantify the numbers of total CD11c⁺ F4/80⁺ (A) and CD11c⁺ F4/80^- (B) cells (n = 5 mice per group; ns, no significant difference <i>vs</i>. Cre- group). (C-D) The VAT-SVF from 10 or 18 wk HFD-fed Cre- and Cre+ mice was analyzed for expression of indicated mRNAs by qRT-PCR (n = 5 mice per group*, <i>p</i> < 0.05 <i>vs</i>. Cre- group; ns, no significant difference <i>vs</i>. Cre- group).</p

Genes involved in ER function, ER stress, and Innate Immunity.

<p>Gene expression profiles were derived from three independent human caseous TB granulomas and averaged, then compared to that of uninvolved lung parenchyma. Shown is the fold upregulation as compared to the control (<i>P</i><0.05). All genes listed under no change did not reach statistical significance.</p

ER stress-induced genes are upregulated in human TB granulomas.

<p>RNA isolated from caseous granulomas by laser capture microdissection from 3 TB patients was subjected to microarray analysis. All genes in the database were ranked and a hierarchical list of each gene in relative transcript abundance was created. Shown is a comparison of ER resident genes that that have not been shown to be regulated by ER stress, common innate immune receptors such as scavenger receptors, TLRs, and macrophage markers, and genes known to participate in the Unfolded Protein Response or ER stress pathway. Error bars represent the standard deviation of caseous granulomas from three independent patients compared in the microarray. Genes represented by a red bars had a ranking above 10 and were represented in the top third of all genes in relative transcript abundance, and the genes represented by green bars fell below that threshold.</p

Increased apoptosis in CHOP-positive regions of human TB granulomas.

<p>Lung sections from TB patients containing granulomas were stained for CHOP (green), TUNEL (red), and nuclei (DAPI; blue). Patients 1 and 2 were positive for granulomas and the negative control was uninvolved lung parenchyma from a TB patient. <b>A.</b> CHOP staining (green) is seen around the central area of caseation (bar represents 100 µm). <b>B.</b> High power magnification shows TUNEL staining in areas that are also positive for CHOP and the DNA stain DAPI (red arrows, bar represents 100 µm). <b>C.</b> Quantification of the percent of CHOP-positive cells from 4 random fields of view. The number of CHOP-positive cells were expressed as a percent of total DAPI-positive cells in each field (n = 4 fields/patient). <b>D.</b> Quantification of the percent of TUNEL-positive cells from 4 random fields of view. The number of cells positive for both TUNEL and DAPI were expressed as a percent of total DAPI-positive cells in each field (n = 4 fields/patient). <b>E.</b> Quantification of the percent of total TUNEL-positive cells that express CHOP. The number of cells positive for TUNEL, DAPI, and CHOP were expressed as a percent of total number of TUNEL-positive cells in each field (n = 4 fields/patient). <b>F.</b> Quantification of the percent TUNEL-positivity in the CHOP expressing population. The number of cells positive for TUNEL, DAPI, and CHOP were expressed as a percent of total number of CHOP-positive cells in each field (n = 4 fields/patient). Differences between values with symbols and no symbols, or between values with different symbols, are statistically significant by ANOVA followed by post-hoc Student-Newman-Keuls test (<i>P</i><0.05).</p

Induction of ER stress markers in human caseous TB granulomas.

<p><b>A and B.</b> Lung sections from TB patients containing granulomas were stained by immunohistochemistry using an antibody against CHOP, normal IgG control, ATF3, and the macrophage marker CD68. Slides were counter-stained with hematoxylin. <b>A.</b> CHOP staining (brown), but not the IgG control, is seen around the central area of caseation (bar represents 100 µm). <b>B.</b> Low and high power magnification shows CHOP staining in areas that are also positive for ATF3 and CD68 (hatched box). The bar in the upper panel represents 1 mm while the bar in the lower panel represents 100 µm. Also shown are multinucleated giant cells surrounding the granuloma also positive for CHOP, ATF3, and CD68 (red arrows).</p

Targeted Interleukin-10 Nanotherapeutics Developed with a Microfluidic Chip Enhance Resolution of Inflammation in Advanced Atherosclerosis

Inflammation is an essential protective biological response involving a coordinated cascade of signals between cytokines and immune signaling molecules that facilitate return to tissue homeostasis after acute injury or infection. However, inflammation is not effectively resolved in chronic inflammatory diseases such as atherosclerosis and can lead to tissue damage and exacerbation of the underlying condition. Therapeutics that dampen inflammation and enhance resolution are currently of considerable interest, in particular those that temper inflammation with minimal host collateral damage. Here we present the development and efficacy investigations of controlled-release polymeric nanoparticles incorporating the anti-inflammatory cytokine interleukin 10 (IL-10) for targeted delivery to atherosclerotic plaques. Nanoparticles were nanoengineered <i>via</i> self-assembly of biodegradable polyester polymers by nanoprecipitation using a rapid micromixer chip capable of producing nanoparticles with retained IL-10 bioactivity post-exposure to organic solvent. A systematic combinatorial approach was taken to screen nanoparticles, resulting in an optimal bioactive formulation from <i>in vitro</i> and <i>ex vivo</i> studies. The most potent nanoparticle termed Col-IV IL-10 NP22 significantly tempered acute inflammation in a self-limited peritonitis model and was shown to be more potent than native IL-10. Furthermore, the Col-IV IL-10 nanoparticles prevented vulnerable plaque formation by increasing fibrous cap thickness and decreasing necrotic cores in advanced lesions of high fat-fed LDLr^–/– mice. These results demonstrate the efficacy and pro-resolving potential of this engineered nanoparticle for controlled delivery of the potent IL-10 cytokine for the treatment of atherosclerosis