Jun Muto

NLRP3/Cryopyrin Is Necessary for Interleukin-1β (IL-1β) Release in Response to Hyaluronan, an Endogenous Trigger of Inflammation in Response to Injury

Inflammation under sterile conditions is a key event in autoimmunity and following trauma. Hyaluronan, a glycosaminoglycan released from the extracellular matrix after injury, acts as an endogenous signal of trauma and can trigger chemokine release in injured tissue. Here, we investigated whether NLRP3/cryopyrin, a component of the inflammasome, participates in the inflammatory response to injury or the cytokine response to hyaluronan. Mice with a targeted deletion in cryopyrin showed a normal increase in Cxcl2 in response to sterile injuries but had decreased inflammation and release of interleukin-1β (IL-1β). Similarly, the addition of hyaluronan to macrophages derived from cryopyrin-deficient mice increased release of Cxcl2 but did not increase IL-1β release. To define the mechanism of hyaluronan-mediated activation of cryopyrin, elements of the hyaluronan recognition process were studied in detail. IL-1β release was inhibited in peritoneal macrophages derived from CD44-deficient mice, in an MH-S macrophage cell line treated with antibodies to CD44, or by inhibitors of lysosome function. The requirement for CD44 binding and hyaluronan internalization could be bypassed by intracellular administration of hyaluronan oligosaccharides (10–18-mer) in lipopolysaccharide-primed macrophages. Therefore, the action of CD44 and subsequent hyaluronan catabolism trigger the intracellular cryopyrin → IL-1β pathway. These findings support the hypothesis that hyaluronan works through IL-1β and the cryopyrin system to signal sterile inflammation.

Ultraviolet radiation damages self noncoding RNA and is detected by TLR3

Exposure to ultraviolet B (UVB) radiation from the sun can result in sunburn, premature aging and carcinogenesis, but the mechanism responsible for acute inflammation of the skin is not well understood. Here we show that RNA is released from keratinocytes after UVB exposure and that this stimulates production of the inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) from nonirradiated keratinocytes and peripheral blood mononuclear cells (PBMCs). Whole-transcriptome sequencing revealed that UVB irradiation of keratinocytes induced alterations in the double-stranded domains of some noncoding RNAs. We found that this UVB-damaged RNA was sufficient to induce cytokine production from nonirradiated cells, as UVB irradiation of a purified noncoding RNA (U1 RNA) reproduced the same response as the one we observed to UVB-damaged keratinocytes. The responses to both UVB-damaged self-RNAs and UVB-damaged keratinocytes were dependent on Toll-like receptor 3 (TLR3) and Toll-like receptor adaptor molecule 1 (TRIF). In response to UVB exposure, Tlr3−/− mice did not upregulate TNF-α in the skin. Moreover, TLR3 was also necessary for UVB-radiation–induced immune suppression. These findings establish that UVB damage is detected by TLR3 and that self-RNA is a damage-associated molecular pattern that serves as an endogenous signal of solar injury.

Staphylococcus epidermidis Antimicrobial δ-Toxin (Phenol-Soluble Modulin-γ) Cooperates with Host Antimicrobial Peptides to Kill Group A Streptococcus

Antimicrobial peptides play an important role in host defense against pathogens. Recently, phenol-soluble modulins (PSMs) from Staphylococcus epidermidis (S. epidermidis) were shown to interact with lipid membranes, form complexes, and exert antimicrobial activity. Based on the abundance and innocuity of the cutaneous resident S. epidermidis, we hypothesized that their PSMs contribute to host defense. Here we show that S. epidermidis δ-toxin (PSMγ) is normally present in the epidermis and sparsely in the dermis of human skin using immunohistochemistry. Synthetic δ-toxin interacted with neutrophil extracellular traps (NETs) and colocalized with cathelicidin while also inducing NET formation in human neutrophils. In antimicrobial assays against Group A Streptococcus (GAS), δ-toxin cooperated with CRAMP, hBD2, and hBD3. In whole blood, addition of δ-toxin exerted a bacteriostatic effect on GAS, and in NETs, δ-toxin increased their killing capacity against this pathogen. Coimmunoprecipitation and tryptophan spectroscopy demonstrated direct binding of δ-toxin to host antimicrobial peptides LL-37, CRAMP, hBD2, and hBD3. Finally, in a mouse wound model, GAS survival was reduced (along with Mip-2 cytokine levels) when the wounds were pretreated with δ-toxin. Thus, these data suggest that S. epidermidis–derived δ-toxin cooperates with the host-derived antimicrobial peptides in the innate immune system to reduce survival of an important human bacterial pathogen.

Engagement of CD44 by hyaluronan suppresses TLR4 signaling and the septic response to LPS.

Fragments of hyaluronan released after injury bind and activate TLR4 in a complex with CD44. Here we investigated if the recognition of hyaluronan by CD44 and TLR4 alters lipopolysaccharide (LPS) responsiveness and thus could alter the septic response. In contrast to mice injected with LPS, mice exposed to hyaluronan prior to LPS had greatly decreased serum IL-6 and TNFalpha and were protected from symptoms of sepsis. The protective effect of HA was not seen in Cd44(-/-) mice. Consistent with our findings in vivo, addition of hyaluronan to macrophages before LPS exposure significantly decreased the release of IL-6 and TNFalpha and this effect was not seen in macrophages from Cd44(-/-) mice. Investigation of the mechanism responsible for inhibition of LPS activation showed hyaluronan treatment resulted in an increase in peritoneal macrophage A20 mRNA expression, and that this was significantly reduced in macrophages from Cd44(-/-) mice and Tlr4(-/-) mice. Suppression of the A20 response with siRNA inhibited the ability of hyaluronan to protect against the cytokine response to LPS. Therefore, our results show that hyaluronan acts through TLR4, CD44 and A20 to stimulate a unique cellular response that can protect against the septic response to LPS.

Hyaluronan digestion controls DC migration from the skin

The breakdown and release of hyaluronan (HA) from the extracellular matrix has been hypothesized to act as an endogenous signal of injury. To test this hypothesis, we generated mice that conditionally overexpressed human hyaluronidase 1 (HYAL1). Mice expressing HYAL1 in skin either during early development or by inducible transient expression exhibited extensive HA degradation, yet displayed no evidence of spontaneous inflammation. Further, HYAL1 expression activated migration and promoted loss of DCs from the skin. We subsequently determined that induction of HYAL1 expression prior to topical antigen application resulted in a lack of an antigenic response due to the depletion of DCs from the skin. In contrast, induction of HYAL1 expression concurrent with antigen exposure accelerated allergic sensitization. Administration of HA tetrasaccharides, before or simultaneously with antigen application, recapitulated phenotypes observed in HYAL1-expressing animals, suggesting that the generation of small HA fragments, rather than the loss of large HA molecules, promotes DC migration and subsequent modification of allergic responses. Furthermore, mice lacking TLR4 did not exhibit HA-associated phenotypes, indicating that TLR4 mediates these responses. This study provides direct evidence that HA breakdown controls the capacity of the skin to present antigen. These events may influence DC function in injury or disease and have potential to be exploited therapeutically for modification of allergic responses.

Hyaluronic Acid Oligosaccharides Suppress TLR3-Dependent Cytokine Expression in a TLR4-Dependent Manner

The release of endogenous molecules from the skin after injury has been proposed to influence inflammation. Recent studies have found that pro-inflammatory signals can be generated by damaged endogenous self-RNA, and this event is detected by TLR3. Conversely, release of endogenous fragments of hyaluronic acid (HA) after injury has been proposed to inhibit LPS induced inflammation driven by TLR4. In this study we investigated if HA oligomers could also influence inflammation mediated by TLR3. A tetramer form of HA (oligo-HA) was added to MH-S cells (mouse alveolar macrophage cell line) that were then activated by poly(I:C). ELISA analysis of culture supernatants showed that the presence of oligo-HA suppressed the poly(I:C) induced release of IL-6 and TNFα. IL-6 mRNA expression was also suppressed as measured by quantitative RT-PCR. To determine the mechanism of action for oligo-HA to inhibit poly(I:C), macrophages derived from wild-type (WT), Tlr2−/− or Tlr4−/− mice were treated with oligo-HA and poly(I:C). Similar to WT cells, Tlr2−/− macrophages were inhibited by oligo-HA and retained suppression of cytokine release. In contrast, Tlr4−/− macrophages lost the capacity to be suppressed by oligo-HA. An increase in Traf1 (TLR negative regulator) mRNA was observed after oligo-HA treatment of WT but not in Tlr4−/− macrophages, and oligo-HA did not suppress cytokine responsiveness in Traf1−/− macrophages. These results show that oligo-HA acts through TLR4 and TRAF1 to inhibit TLR3-dependent inflammation. This observation illustrates the complex immunomodulatory action of endogenous products released after injury.

Hyaluronan Breakdown Contributes to Immune Defense against Group A Streptococcus

Background: The role of hyaluronan catabolism in group A Streptococcus (GAS) infection has not been studied. Results: Hyaluronan size differentially influenced GAS infection in vitro and in mice. Conclusion: Digestion of hyaluronan derived from either the bacterium or host enhances host immune defense. Significance: Digestion of hyaluronan during GAS infection may be a previously unrecognized mechanism for host defense. Group A Streptococcus (GAS) commonly infects human skin and occasionally causes severe and life-threatening invasive diseases. The hyaluronan (HA) capsule of GAS has been proposed to protect GAS from host defense by mimicking endogenous HA, a large and abundant glycosaminoglycan in the skin. However, HA is degraded during tissue injury, and the functions of short-chain HA that is generated during infection have not been studied. To examine the impact of the molecular mass of HA on GAS infection, we established infection models in vitro and in vivo in which the size of HA was defined by enzymatic digestion or custom synthesis. We discovered that conversion of high molecular mass HA to low molecular mass HA facilitated GAS phagocytosis by macrophages and limited the severity of infection in mice. In contrast, native high molecular mass HA significantly impaired internalization by macrophages and increased GAS survival in murine blood. Thus, our data demonstrate that GAS virulence can be influenced by the size of HA derived from both the bacterium and host and suggest that high molecular mass HA facilitates GAS deep tissue infections, whereas the generation of short-chain HA can be protective.

Exogenous Addition of a C-Xylopyranoside Derivative Stimulates Keratinocyte Dermatan Sulfate Synthesis and Promotes Migration

As C-Xyloside has been suggested to be an initiator of glycosaminoglycan (GAG) synthesis, and GAGs such as Dermatan sulfate (DS) are potent enhancers of fibroblast growth factor (FGF) - 10 action, we investigated if a C-Xylopyranoside derivative, (C-β-D-xylopyranoside-2-hydroxy-propane, C-Xyloside), could promote DS production by cultured normal human keratinocytes, how this occurs and if C-Xyloside could also stimulate FGF-dependent cell migration and proliferation. C-Xyloside-treated keratinocytes greatly increased secretion of total sulfated GAGs. Majority of the induced GAG was chondroitin sulfate/dermatan sulfate (CS/DS) of which the major secreted GAG was DS. Cells lacking xylosyltransferase enzymatic activity demonstrated that C-Xyloside was able to stimulate GAG synthesis without addition to core proteins. Consistent with the observed increase in DS, keratinocytes treated with C-Xyloside showed enhanced migration in response to FGF-10 and secreted into their culture media GAGs that promoted FGF-10-dependent cellular proliferation. These results indicate that C-Xyloside may enhance epithelial repair by serving as an initiator of DS synthesis.

Tissue damage drives co-localization of NF-κB, Smad3, and Nrf2 to direct Rev-erb sensitive wound repair in mouse macrophages

Although macrophages can be polarized to distinct phenotypes in vitro with individual ligands, in vivo they encounter multiple signals that control their varied functions in homeostasis, immunity, and disease. Here, we identify roles of Rev-erb nuclear receptors in regulating responses of mouse macrophages to complex tissue damage signals and wound repair. Rather than reinforcing a specific program of macrophage polarization, Rev-erbs repress subsets of genes that are activated by TLR ligands, IL4, TGFβ, and damage-associated molecular patterns (DAMPS). Unexpectedly, a complex damage signal promotes co-localization of NF-κB, Smad3, and Nrf2 at Rev-erb-sensitive enhancers and drives expression of genes characteristic of multiple polarization states in the same cells. Rev-erb-sensitive enhancers thereby integrate multiple damage-activated signaling pathways to promote a wound repair phenotype. DOI: http://dx.doi.org/10.7554/eLife.13024.001

Case of psoriatic erythroderma induced by the discontinuation of the chronic use of topical steroid after dialysis initiation and successfully treated with secukinumab

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