TFAM and CpGA DNA Induce a Splenocyte Proinflammatory Immune Response through PI3K and NF-κB Signaling.

<p>The presented data was derived from at least 5 independent experiments. TFAM (5 µg/ml) + CpGA DNA (0.3 µM)-induced Flt3L-expanded splenocyte (1 × 10⁶ cells/ml) TNFα release was completely blocked 24 hours post-treatment following pre-treatment (30 minutes) with inhibitors of PI3K (LY294002, 5 µM) and NF-κB (BAY 11-7085, 5 µM) signaling (*<i>p</i> < 0.01, relative to no treatment; † <i>p</i> < 0.01, compared to the TFAM + CpGA treatment group).</p

Amplification of CpGA DNA-provoked Splenocyte TNFα Release by TFAM Involves RAGE and TLR9 but Not FPR.

<p>The presented data was derived from at least 5 independent experiments. (A) 24 hours post-treatment, TFAM (5 µg/ml) increased CpGA DNA (0.3 µM)-induced Flt3L-expanded mouse splenocyte (1 × 10⁶ cells/ml) TNFα release. This effect was dramatically attenuated through RAGE (sRAGE, 20 µg/ml) or TLR9 (G-ODN, 10 µM) inhibition 30 minutes pre-treatment. In addition, splenocytes from RAGE -/- mice demonstrated significantly diminished TNFα release in response to TFAM ± CpGA DNA treatment. (B) Similarly, exposure of Flt3L-expanded splenocytes (1 × 10⁶ cells/ml) to immunoprecipitated native TFAM complexed with mtDNA (IP TFAM, 0.5 µg/ml) derived from necrotic cells yielded marked release of TNFα by 24 hours post-treatment which was significantly reduced when treating corresponding splenocytes from RAGE -/- mice. (C) Pre-treatment (30 minutes) with FPR antagonists [CsH (1 µM), BOC (10 µM) or WRW4 (20 µg/ml)] had no effect upon TFAM (5 µg/ml) + CpGA DNA (0.3 µM)-induced splenocyte TNFα release 24 hours post-treatment. Likewise, 24 hours after treatment, FPR agonists [fMLP (5 µg/ml), ND6 (5 µg/ml)] did not stimulate TNFα release (*<i>p</i> < 0.01, compared to no treatment; ^†<i>p</i> < 0.01, relative to the wild type TFAM + CpGA treatment group).</p

Schematic Representation of Splenic Dendritic Cell Activation by Necrotic Cells.

<p>Mitochondrial DNA (mtDNA), when released from necrotic cells, remains associated with TFAM. As in Type I interferon production, TFAM augments the proinflammatory immune response to mtDNA by associating with RAGE (and related heparin sulfate moieties) and TLR9, respectively, to promote endosomal processing, including endosomal receptor (TLR9) recycling by ECE-1, and signal transduction via PI3K/Akt and ERK pathway activation to induce splenic dendritic cell TNFα gene transcription and protein translation and release.</p

TFAM Augments CpGA DNA-induced Splenocyte TNFα Release which Is Highly Dependent upon the Presence of Plasmacytoid Dendritic Cells.

<p>The presented data was derived from at least 5 independent experiments. (A) As previously observed for other cytokines in different cell preparations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072354#B2" target="_blank">2</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072354#B3" target="_blank">3</a>], native mitochondrial protein (5 µg/ml) induced significant TNFα release from cultured mouse Flt3L-expanded splenocytes (1 × 10⁶ cells/ml) 24 hours post-treatment. CpGA DNA (0.3 µM), which is akin to that naturally associated with mitochondrial protein, promoted splenocyte TNFα release which was significantly amplified by co-incubation with recombinant human TFAM (5 µg/ml) despite that having no effect when treated alone. Compared to splenocytes obtained from a normal spleen, TNFα release was markedly increased following all treatments when the constituent pDC population was expanded by Flt3L over-expression as employed in the present study. (B) Selective removal of pDCs from matching Flt3L-expanded splenocyte preparations notably reduced the TNFα release induced by CpGA DNA (0.3 µM) ± TFAM (5 µg/ml) 24 hours post-treatment. (C) pDCs (1 × 10⁶ cells/ml) isolated from similar mouse splenocyte preparations demonstrated comparably minimal yet significant TNFα release 24 hours after CpGA DNA (0.3 µM) ± TFAM (5 µg/ml) treatment (*<i>p</i> < 0.01, relative to no treatment; ^†<i>p</i> < 0.01, compared to CpGA DNA treatment alone, and ^‡<i>p</i> < 0.01, relative to matching normal splenocyte treatments).</p

Enhancement by TFAM of the CpGA DNA-induced Splenocyte Proinflammatory Immune Response Results from Charge-Dependent Recognition of TFAM and Is Equipotent for Both TFAM DNA-binding Subunit Proteins, Box 1 and Box 2.

<p>The presented data was derived from at least 5 independent experiments. (A) Pre-treatment (30 minutes) with heparin sodium (1-100 U/ml) or heparin lyase (heparinase) II or III (5 mU/ml) dramatically suppressed TFAM (5 µg/ml) + CpGA DNA (0.3 µM)-induced Flt3L-expanded splenocyte (1 × 10⁶ cells/ml) TNFα release 24 hours post-treatment. Heparin demonstrated significant inhibition in a dose-dependent manner (*<i>p</i> < 0.01, compared to no treatment; † <i>p</i> < 0.01, relative to the TFAM + CpGA treatment group). (B) Though not as potent at augmenting CpGA DNA (0.3 µM)-induced splenocyte (1 × 10⁶ cells/ml) TNFα release as full-length recombinant TFAM (5 µg/ml) 24 hours post-treatment, recombinant TFAM Box 1 and Box 2 (5 µg/ml) each demonstrated significant amplification of CpGA DNA-induced TNFα release (*<i>p</i> < 0.01, compared to no treatment; † <i>p</i> < 0.01, relative to CpGA DNA treatment alone, and ‡<i>p</i> < 0.01, compared to CpGA DNA alone and the TFAM + CpGA treatment group).</p

Elevated splenocyte IRAK-M expression in response to repeated TLR-4 or TLR-9 ligand exposures is suppressed by IL-7 pre-treatment.

<p>The presented data is representative of at least 3 independent experiments. (A) Representative photomicrograph of a Western blot demonstrating the changes observed in IRAK-M expression 48 hours after successive 24-hour repeated ligand exposures (with or without a 30 minute IL-7 pre-treatment). (B) Relative band density of splenocyte IRAK-M expression was dramatically elevated for both successive treatment combinations and notably reduced with intervening IL-7 pre-treatment [*<i>p</i> < 0.05, relative to the untreated (Vehicle, Vehicle) control group, and ^†<i>p</i> < 0.05, compared to the corresponding LPS treatment combination in the absence of IL-7 (LPS, LPS)].</p

Cross-tolerance to splenocyte cytokine release results from Sequential exposures to LPS followed by CpGA DNA.

<p>The presented data was derived from at least 4 independent experiments, each in triplicate. (A) Flt3L-expanded splenocyte (1 × 10⁶ cells/ml) TNFα release was dramatically diminished 24 hours following treatment with CpGA DNA (1 μM) after a prior 24-hour exposure to LPS (10 ng/ml). (B) Similarly, following a previous 24-hour exposure to LPS, splenocyte IFNγ release was significantly suppressed 24 hours after treatment with CpGA DNA. (C) Consistently, splenocyte IL-10 release demonstrated the same pattern of cross-tolerance in response to CpGA DNA at 24 hours post-treatment after an earlier 24-hour exposure to LPS [*<i>p</i> < 0.01, compared to time-matched untreated (Vehicle, Vehicle) controls; ^†<i>p</i> < 0.05, relative to the time-matched CpGA treatment alone group (Vehicle, <u>CpGA</u>), and ^‡<i>p</i> < 0.01, compared to the time-matched untreated (Vehicle, <u>Vehicle</u>) control and the associated LPS treatment group at 24 hours (<u>LPS</u>, Vehicle)].</p

Tolerance and cross-tolerance to splenocyte cytokine release is unaffected by blocking PD-1.

<p>The presented data was derived from at least 3 independent experiments, each in triplicate. Data represents splenocyte cytokine release 48 hours after successive 24-hour exposures to the ligand treatment combinations detailed on the X-axis. (A) Pre-treatment (30 minutes) of Flt3L-expanded splenocytes (1 × 10⁶ cells/ml) with an mouse anti-PD-1 blocking antibody (or corresponding isotypic control antibody, PD-1C) (5 μg/ml) prior to the secondary 24-hour ligand treatment had no effect upon resultant TNFα release regardless of the treatment combination employed. (B) Similarly, anti-PD-1 pre-treatment did not alter resultant splenocyte IFNγ release for any of the treatment combinations used. (C) Consistently, resultant splenocyte IL-10 release was unaffected for all treatment combinations by anti-PD-1 pre-treatment.</p

IRAK-M-/- splenocytes demonstrate diminished tolerance and cross-tolerance through improved responsiveness to subsequent ligand exposures.

<p>The presented data was derived from at least 3 independent experiments, each in triplicate. Data represents splenocyte cytokine release [as a percent of secondary ligand exposure in matching wild-type (WT) splenocytes] 48 hours after successive 24-hour exposures to the ligand treatment combinations detailed on the X-axis. (A) Following a 24-hour exposure to CpGA DNA (1 μM), Flt3L-expanded mouse IRAK-M-/- splenocytes (1 × 10⁶ cells/ml) showed significantly improved responsiveness to subsequent CpGA DNA-induced TNFα release at 24 hours post-treatment which was further augmented by IL-7 (25 ng/ml) pre-treatment (30 minutes). (B) Regardless of the treatment combination employed, tolerance and cross-tolerance to IRAK-M-/- splenocyte IFNγ release in response to the secondary ligand exposure was significantly attenuated. This observation was unaffected by IL-7 pre-treatment. (C) IRAK-M-/- splenocytes demonstrated no enhancement in IL-10 responsiveness to the secondary ligand exposure regardless of IL-7 pre-treatment [*<i>p</i> < 0.01, relative to the matching wild-type secondary treatment (white bar), and ^†<i>p</i> < 0.01, compared to the matching CpGA DNA secondary treatment in the absence of IL-7 (black bar)].</p

Repeated exposure to TLR-4 ligand, LPS, induces tolerance to splenocyte pro-inflammatory cytokine release.

<p>The presented data was derived from at least 4 independent experiments, each in triplicate. (A) LPS (10 ng/ml) induced significant TNFα release from cultured Flt3L-expanded mouse splenocytes (1 × 10⁶ cells/ml) 24 hours post-treatment. In response to a subsequent LPS treatment, TNFα release was dramatically reduced 24 hours later. (B) Similarly, significant splenocyte IFNγ release was observed 24 hours following LPS treatment. IFNγ release was markedly decreased 24 hours after a successive LPS treatment. This release, however, was very similar to the sustained IFNγ release observed over the subsequent 24-hour period in the absence of follow-up treatment (LPS, <u>Vehicle</u>). (C) Splenocytes failed to demonstrate any IL-10 release in response to LPS treatment [*<i>p</i> < 0.01, relative to time-matched untreated (Vehicle, Vehicle) controls; ^†<i>p</i> < 0.01, compared to the associated LPS treatment group at 24 hours (<u>LPS</u>, LPS) and the time-matched LPS treatment alone group (Vehicle, <u>LPS</u>), and ^‡<i>p</i> < 0.01, relative to the time-matched untreated (Vehicle, <u>Vehicle</u>) control and the associated LPS treatment group at 24 hours (<u>LPS</u>, Vehicle)].</p