Background: Apoptotic cell recognition triggers profound immunosuppressive responses; relevant recognition determinants are uncharacterized. Results: Surface exposure of glycolytic enzymes is a common early apoptotic event. Conclusion: Externalized glycolytic enzyme molecules are novel apoptotic biomarkers and candidate immunomodulatory/ recognition determinants. Significance: Apoptotic glycolytic enzyme externalization explicates plasminogen binding to mammalian cells and potential mechanisms of immune privilege by commensal bacteria and pathogens.

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Background: Dysregulated Toll-like receptor (TLR) signaling is a hallmark of endotoxin-tolerized macrophages in immunosuppression stages of sepsis but Pellino-1 involvement is unknown. Results: Endotoxin tolerization suppresses LPS-induced Pellino-1, Pellino-1 potentiates TLR2/4-driven NF-B, cytokines and K63-linked IRAK1, TBK1, TAK1, and TRAF6 polyubiquitination. Conclusion: Pellino-1 potentiates TLR2/4 signaling and is decreased by endotoxin tolerization. Significance: Uncovering mechanisms of endotoxin tolerance is critical to understand sepsis-associated immunosuppression.

Development of endotoxin tolerance in macrophages during sepsis reprograms Toll-like receptor 4 signaling to inhibit proinflammatory cytokines without suppressing anti-inflammatory and antimicrobial mediators and protects the host from excessive inflammation and tissue damage. However, endotoxin tolerance renders septic patients immunocompromised and unable to control secondary infections. Although previous studies have revealed the importance of several negative regulators of Toll-like receptor signaling in endotoxin tolerance, the role of Pellino proteins has not been addressed. The present report shows that the induction of endotoxin tolerance in vivo in mice and in vitro in human monocytes and THP-1 and MonoMac-6 macrophages increases the expression of Pellino-3. Overexpression of Pellino-3 in human embryonic kidney 293/Toll-like receptor 2 or 293/Toll-like receptor 4/myeloid differentiation factor-2 cells inhibited Toll-like receptor 2/4-mediated activation of nuclear factor-κB and induction of CXCL-8 mRNA, and Pellino-3 ablation increased these responses. Pellino-3-deficient THP-1 cells had elevated Toll-like receptor 2/4-driven tumor necrosis factor-α, interleukin-6 mRNA, and Toll-like receptor 4-driven CCL5 gene expression in response to Toll-like receptor agonists and heat-killed Escherichia coli and Staphylococcus aureus, cytokines controlled by the MyD88 and Toll-interleukin-1R domain-containing protein inducing interferon-β-mediated pathways, respectively. In addition, deficiency in Pellino-3 slightly increased phagocytosis of heat-killed bacteria. Transfected Pellino-3 inhibited nuclear factor-κB activation driven by overexpression of MyD88, TIR domain-containing adapter inducing interferon-β, interleukin-1R-associated kinase-1, and tumor necrosis factor receptor activator of nuclear factor-κB-binding kinase-1, TGF-β-activated kinase 1, and tumor necrosis factor receptor-associated factor-6, and inhibited interleukin-1R-associated kinase 1 modifications and tumor necrosis factor receptor activator of nuclear factor-κB-binding kinase 1 phosphorylation. Finally, Pellino-3 ablation in THP-1 decreased the extent of endotoxin tolerization. Thus, Pellino-3 is involved in endotoxin tolerance and functions as a negative regulator of Toll-like receptor 2/4 signaling.

Toll-like receptor 2 (TLR2) plays a critical role in host defenses against mycobacterial infections. The R753Q TLR2 polymorphism has been associated with increased incidence of tuberculosis and infections with non-tuberculous mycobacteria in human populations, but the mechanisms by which this polymorphism affects TLR2 signaling are unclear. In this study, we determined the impact of the R753Q TLR2 polymorphism on macrophage sensing of Upon infection with, macrophages from knock-in mice harboring R753Q TLR2 expressed lower levels of TNF-α, IL-1β, IL-6, and IL-10 compared with cells from WT mice, but both R753Q TLR2- and WT-derived macrophages exhibited comparable bacterial burdens. The decreased cytokine responses in R753Q TLR2-expressing macrophages were accompanied by impaired phosphorylation of IL-1R-associated kinase 1 (IRAK-1), p38, ERK1/2 MAPKs, and p65 NF-κB, suggesting that the R753Q TLR2 polymorphism alters the functions of the myeloid differentiation primary response protein 88 (MyD88)-IRAK-dependent signaling axis. Supporting this notion, HEK293 cells stably transfected with YFP-tagged R753Q TLR2 displayed reduced recruitment of MyD88 to TLR2, decreased NF-κB activation, and impaired IL-8 expression upon exposure to Collectively, our results indicate that the R753Q polymorphism alters TLR2 signaling competence, leading to impaired MyD88-TLR2 assembly, reduced phosphorylation of IRAK-1, diminished activation of MAPKs and NF-κB, and deficient induction of cytokines in macrophages infected with.

SummaryImmunosenescence is a state of unbalanced immune responsiveness, characterized by a diverse repertoire of seemingly discreet and paradoxical alterations in all aspects of immunity arising in an aging‐associated manner. We asked whether aging‐associated alterations in the ability of apoptotic cells to elicit immunomodulatory responses (innate apoptotic immunity; IAI) or in IAI responses themselves might underlie the confounding aging‐associated anomalies of immunosenescence. We explored this question by examining, as a function of animal age, responsiveness of murine macrophages on the single cell level. We monitored the expression of pro‐ and anti‐inflammatory cytokines cytofluorimetrically in response to pro‐inflammatory Toll‐like receptor (TLR) stimulation and anti‐inflammatory treatment with apoptotic cells. While we found no alterations with age in the potency of apoptotic cells or in the initiation and magnitude of IAI responses, we did identify a cell‐intrinsic deficiency in anti‐inflammatory IAI response termination linked with age and preceding manifestations of immunosenescence. Further, we found that an aging‐associated deficiency in response termination also is evident following TLR stimulation. These surprising observations reveal that a loss of homeostatic immune control with animal age results from the dysregulation of response termination (as distinct from response initiation) and is exerted on the level of transcription. We suggest that, with advancing age, cells become locked into relatively longer‐lived response states. Aging‐associated immune dysfunctions may reflect a diminution in the cellular nimbleness of immune responsiveness.

IL-1 receptor-associated kinase (IRAK) 4 is a central enzyme of the TLR pathways. This study tested the hypothesis that IRAK4 kinase activity is prerequisite for regulating innate immunity during infections with intracellular bacteria. To this end, we analyzed responses of macrophages obtained from mice expressing wild-type (WT) IRAK4 or its kinase-inactive K213M mutant (IRAK4 ) upon infection with intracellular bacteria Listeria monocytogenes or Mycobacterium smegmatis. In contrast to robust induction of cytokines by macrophages expressing kinase-sufficient IRAK4, IRAK4 macrophages expressed decreased TNF-α, IL-6, IL-1β, and C-C motif chemokine ligand 5 upon infection with L. monocytogenes or M. smegmatis. Bacterial infection of IRAK4 macrophages led to attenuated activation of IRAK1, MAPKs and NF-κB, impaired induction of inducible NO synthase mRNA and secretion of NO, but resulted in elevated microbial burdens. Compared with WT animals, systemic infection of IRAK4 mice with M. smegmatis or L. monocytogenes resulted in decreased levels of serum IL-6 and CXCL-1 but increased bacterial burdens in the spleen and liver. Thus, a loss of IRAK4 kinase activity underlies deficient cytokine and microbicidal responses during infection with intracellular bacteria L. monocytogenes or M. smegmatis via impaired activation of IRAK1, MAPKs, and NF-κB but increases bacterial burdens, correlating with decreased induction of NO.

Intraurethral inoculation of mice with uropathogenic E. coli (CP1) results in prostate inflammation, fibrosis, and urinary dysfunction, recapitulating some but not all of the pathognomonic clinical features associated with benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS). In both patients with LUTS and in CP1-infected mice, we observed increased numbers and activation of mast cells and elevated levels of prostate fibrosis. Therapeutic inhibition of mast cells using a combination of mast cell stabilizer (MCS), cromolyn sodium, and the histamine 1 receptor antagonist (H1RA), cetirizine di-hydrochloride, in the mouse model resulted in reduced mast cell activation in the prostate and significant alleviation of urinary dysfunction. Treated mice showed reduced prostate fibrosis, less infiltration of immune cells, and decreased inflammation. In addition, as opposed to symptomatic CP1-infected mice, treated mice showed reduced myosin light chain (MLC)-2 phosphorylation, a marker of prostate smooth muscle contraction. These results show that mast cells play a critical role in the pathophysiology of urinary dysfunction and may be an important therapeutic target for men with BPH/LUTS.