<i>C. albicans</i>–mediated NLRP3 inflammasome activation.

<p>Upon encountering <i>C. albicans</i>, pattern recognition receptors (PRR) on the macrophage, such as TLR2 and Dectin 1 and 2, activate NF-κB, leading to the transcription and translation of NLRP3 and pro-IL-1β. Phagocytosis of <i>C. albicans</i> yeast forms triggers hyphal formation which may result in lysosomal rupture. NLRP3 inflammasome activation is then triggered through an as-yet-undefined mechanism. Although morphogenesis appears to be necessary for inflammasome activation, it is not sufficient. Activation of the NLRP3 inflammasome results in activation of the cysteine protease caspase-1, which mediates the processing and secretion of pro-IL-1β and pro-IL-18. Caspase-1 activation also induces pyroptotic cell death of the macrophage, resulting in cell swelling, DNA fragmentation, and the lytic release of intracellular inflammatory contents. Osmotic lysis of the macrophage during pyroptosis can be inhibited by the addition of extracellular glycine.</p

The ESX-1 secretion system of Mtb mediates induction of apoptotic host cell death.

<p>(A) BMDCs were left uninfected (UI), infected with wild-type Mtb (Mtb) or the <i>esxA</i> deletion mutant (ΔesxA) for 4h at MOI of 10, washed and incubated for an additional 24 h. Cells were harvested and stained using the TUNEL assay. The percent of TUNEL positive cells was determined by flow cytometry. (B) BMDCs were infected as described for (A) and then chased for the indicated time periods. The percentage of cells with active caspase-3/7 was detected as described in material and methods. Shown are means and standard deviation of triplicate measurements of one representative experiment out of three.</p

Highly purified CD11c⁺ dendritic cells confirmed the partially caspase-1/11 independent inflammasome activation upon infection with Mtb.

<p>CD11c⁺ sorted BMDCs from wild-type WT and <i>Casp1/11^−/−</i> were left uninfected (UI, black bars), infected with Mtb (white bars) or the ΔesxA mutant (gray bars). After 24h the percentage of TUNEL positive cells was determined in (A). In (B) the amount of necrosis was determined by quantification of the release of adenylate kinase and normalized to the values obtained in detergent lysed cells (PC, striped bar). In (C) and (D) the same supernatants were analyzed for IL-1β and IL-18 secretion, respectively.</p

The IL-1 receptor is not necessary for Mtb mediated IL-1β secretion and has a minor role in cell death induction.

<p>BMDCs from wild-type (WT) or IL-1RI deficient mice (<i>Il1r1^−/−</i>) were infected with Mtb (white bars) or left uninfected (UI, black bars) and analyzed for IL-1β secretion in (A) and TUNEL staining in (B). Shown are means and standard deviation of triplicate measurements of one representative experiment out of three.</p

The ESX-1 secretion system of Mtb does not affect secretion of proinflammatory cytokines in dendritic cells but is important for complete inflammasome activation.

<p>BMDCs were left uninfected (UI), infected with wild-type Mtb (Mtb) or the esxA deletion mutant (ΔesxA) for 4 h at MOI of 10, washed and incubated for an additional 24 h (A+B) or the indicated timepoints (C+D). (A) The cytokine profile in the supernatants was analyzed using a bead-based immunoassay (black =  uninfected, white =  Mtb, gray =  ΔesxA). (B) and (C) IL-1β secretion was analyzed by ELISA. (D) The percent of cells with activated caspase-1 was determined via flow cytometry using fluorescent caspase-1 substrates (FLICA). (E) Pro-IL-1β protein levels in BMDCs. (F) GFP-labeled bacteria were used to infect BMDCs and rate of infection was determined via flow cytometry. Shown are means and standard deviation of triplicate measurements of one representative experiment out of three. In all figures, the asterisks denote range of p values (* = p<0.05, ** = 0.01>p>0.001,***p<0.001, ns =  not significant ) as determined by one way ANOVA with Tukey's post test.</p

BMDC inflammasome activation upon Mtb infection is dependent upon host cell caspase-1/11, NLRP3 and ASC but not NLRC4 and NOD-2.

<p>BMDCs were derived from wild-type (WT), <i>Casp1/11^−/−</i> and <i>Nlrc4^−/−</i> mice (A), WT, <i>Nlrp3^−/−</i> and <i>Asc^−/−</i> mice (B) or WT and NOD-2 mice (C) and infected with Mtb (black bar), ΔesxA (gray bar), left uninfected (UI, black bar) or treated with LPS and ATP (striped bar) as a positive control. The supernatants were harvested after 24 h and the amount of secreted IL-1β was detected by ELISA. Shown are means and standard deviation of triplicate measurements of one representative experiment out of three.</p

NLRC4-deficiency leads to decreased signaling pathways related to DN pathology.

<p><b>(A)</b>. The mRNA fold change of <i>Mcp1</i>, <i>Ccl3</i>, <i>Ccl4</i>, and <i>Cxcl1</i> in the kidney tissues was determined by real-time PCR, using <i>Gapdh</i> as a reference gene, from the indicated groups. Data is presented as the average fold change compared to control ± s.e.m. n = 3–4. <b>(B)</b>. Western blot showing altered activation of different signaling pathways including NF-κB and JNK pathways in the renal tissues from different groups. Images represent two independent experiments from a total of 3 kidneys within each indicated groups. <b>(C)</b>. The mRNA fold change of <i>Tnfα</i>, <i>Tgfβ</i>, and <i>Ctgf</i> in the renal tissues was determined by real-time PCR, using <i>Gapdh</i> as a reference gene. Data is presented as the average fold change compared to controls ± s.e.m. n = 3. *, <i>P</i><0.05 vs. control WT; #, <i>P</i> <0.05 vs. diabetic WT. n = 3.</p

NLRC4-deficiency ameliorates hyperglycemia and renal morphological changes in mice.

<p><b>A-C</b>. Mice were fed an HFD for 4 weeks then injected with STZ and monitored for 8 weeks. Blood glucose levels (<b>A</b>), body weight changes (<b>B</b>), and urinary albumin to creatinine ratio (ACR) (<b>C</b>) at different time pointes after STZ treatment were measured from normal WT, diabetic WT and diabetic <i>Nlrc4</i>^-/- mice. <b>D</b>. Mice were killed 8 weeks after STZ treatment and the kidneys were harvested. Kidney sections were stained with hematoxylin and Eosin staining (H&E) and PAS staining (magnification×400). *, <i>P</i><0.05 vs. control WT; #, <i>P</i> <0.05 vs. diabetic WT. n = 6 for all these experiments. Scale bar: 100 μm.</p

NLRC4-inflammasome activation in the renal tissues of DN mice.

<p><b>(A)</b> ELISA analysis of IL-1β levels in kidney tissues from different groups including normal WT, diabetic WT and diabetic <i>Nlrc4</i>^-/- mice. Values are means ± s.e.m. n = 3. (<b>B)</b> The mRNA levels of <i>Nlrc4</i> and <i>Casp-1</i> in renal tissues from different groups by real time PCR analysis, using <i>Gapdh</i> as a reference gene. Values are mean fold change compared to control mice ± s.e.m. *, P<0.05 vs. control WT; #, P <0.05 vs. diabetic WT. n = 3 each group. (<b>C)</b> Western blot analyses of cleaved Casp-1 p10 and p20 subunits in renal lysates from different groups. n = 3 each group. (<b>D)</b> Protein densitometry analysis of C to show the processing and activation of pro-casp1 into p20 and p10 subunits. Data represents mean protein densitometry in pixels and normalized to the corresponding Tubulin, quantitated by <i>Image J</i> ± s.e.m. n = 3.</p

Increased expression of NLRC4 and macrophage infiltration in renal tissues of DN patients.

<p><b>(A)</b>: PAS staining showing GBM thickening and mesangial expansion in renal biopsies in DN patients (Aa vs. Ab, Scale Bar: 200 μm). IHC studies revealed increased expression of NLRC4 and CD68 in DN patients (Ac-Af, Scale Bar: 100 μm). <b>(B)</b>: Averaged relative intensity for the staining of NLRC4 (top panel) and CD68 (bottom panel) in kidney biopsies of control versus DN patients. <b>(C and D)</b>: Blood glucose levels and serum creatinine (SCR) in control and DN patients. Values are means ± s.e.m. Con: control patient; DN: diabetic patient. *<i>P</i><0.05.</p