NLRP1 activation by UVB: Shedding light on an enigmatic inflammasome.

In this issue of JEM, Jenster et al. (2022. J. Exp. Med. https://doi.org/10.1084/jem.20220837) investigate how UVB radiation promotes activation of the inflammatory immune sensor NLRP1, and in doing so uncover how NLRP1 recognizes a diverse range of ribotoxic stresses.

ZAKα-driven ribotoxic stress response activates the human NLRP1 inflammasome.

Human NLRP1 (NACHT, LRR, and PYD domain-containing protein 1) is an innate immune sensor predominantly expressed in the skin and airway epithelium. Here, we report that human NLRP1 senses the ultraviolet B (UVB)- and toxin-induced ribotoxic stress response (RSR). Biochemically, RSR leads to the direct hyperphosphorylation of a human-specific disordered linker region of NLRP1 (NLRP1DR) by MAP3K20/ZAKα kinase and its downstream effector, p38. Mutating a single ZAKα phosphorylation site in NLRP1DR abrogates UVB- and ribotoxin-driven pyroptosis in human keratinocytes. Moreover, fusing NLRP1DR to CARD8, which is insensitive to RSR by itself, creates a minimal inflammasome sensor for UVB and ribotoxins. These results provide insight into UVB sensing by human skin keratinocytes, identify several ribotoxins as NLRP1 agonists, and establish inflammasome-driven pyroptosis as an integral component of the RSR.

Investigating the Role of Inflammasome Caspases 1 and 11 in the Acute Radiation Syndrome.

Exposure to high dose radiation causes life-threatening acute and delayed effects. Defining the mechanisms of lethal radiation-induced acute toxicity of gastrointestinal and hematopoietic tissues are critical steps to identify drug targets to mitigate and protect against the acute radiation syndrome (ARS). For example, one rational approach would be to design pharmaceuticals that block cell death pathways to preserve tissue integrity in radiation-sensitive organ systems including the gastrointestinal tract and hematopoietic compartment. A previous study reported that the inflammasome pathway, which mediates inflammatory cell death through pyroptosis, promotes ARS. However, we show that mice lacking the inflammatory executioner caspases, caspase-1 and caspase-11, are not protected from ARS when compared directly to littermates expressing caspase-1 and caspase-11. These results suggest that alternative pathways will need to be targeted by drugs that successfully mitigate and protect against the ARS.

Same pollution sources for climate change might be hyperactivating the NLRP3 inflammasome and exacerbating neuroinflammation and SARS mortality.

We have reviewed a considerable amount of recent scientific papers relating inflammation caused by air pollution with chronic and severe medical conditions. Furthermore, there are evidences relating organ inflammation caused by not only outdoor long-term but also short-term inhaled radioisotopes contained in high polluted air or in household natural radioactive background aerosols, in addition to SARS-COV-2 attached to bioaerosols, which are related with a worst evolution of severe acute respiratory syndrome patients. Reactive oxygen species (ROS) production induced by the interaction with environmental ionizing radiation contained in pollution is pointed out as a critical mechanism that predispose mainly to elder population, but not excluding young subjects, presenting previous chronic conditions of lung inflammation or neuroinflammation, which can lead to the most serious consequences.

Radiation causes tissue damage by dysregulating inflammasome-gasdermin D signaling in both host and transplanted cells.

Radiotherapy is a commonly used conditioning regimen for bone marrow transplantation (BMT). Cytotoxicity limits the use of this life-saving therapy, but the underlying mechanisms remain poorly defined. Here, we use the syngeneic mouse BMT model to test the hypothesis that lethal radiation damages tissues, thereby unleashing signals that indiscriminately activate the inflammasome pathways in host and transplanted cells. We find that a clinically relevant high dose of radiation causes severe damage to bones and the spleen through mechanisms involving the NLRP3 and AIM2 inflammasomes but not the NLRC4 inflammasome. Downstream, we demonstrate that gasdermin D (GSDMD), the common effector of the inflammasomes, is also activated by radiation. Remarkably, protection against the injury induced by deadly ionizing radiation occurs only when NLRP3, AIM2, or GSDMD is lost simultaneously in both the donor and host cell compartments. Thus, this study reveals a continuum of the actions of lethal radiation relayed by the inflammasome-GSDMD axis, initially affecting recipient cells and ultimately harming transplanted cells as they grow in the severely injured and toxic environment. This study also suggests that therapeutic targeting of inflammasome-GSDMD signaling has the potential to prevent the collateral effects of intense radiation regimens.

Hippocampal changes in inflammasomes, apoptosis, and MEMRI after radiation-induced brain injury in juvenile rats.

PURPOSE: The aim of this study was to characterize changes in hippocampal inflammasomes, pyroptosis and apoptosis in juvenile rats after brain irradiation and to assess whether manganese-enhanced magnetic resonance imaging (MEMRI) reflected those changes. MATERIALS AND METHODS: Four-week-old male Sprague-Dawley rats received a whole-brain radiation dose of 15 Gy or 25 Gy. Hippocampal inflammasomes and apoptosis were measured using Western blot analysis at 4 days and 8 weeks after irradiation. MEMRI and magnetic resonance spectroscopy (MRS) were performed at the same time points. RESULTS: Neither the 15 Gy nor 25 Gy group showed changes in the expression of inflammasome proteins absent in melanoma 2 (AIM2), gasdermin-D (GSDMD), nucleotide oligomerization domain-like receptor protein 1 (NLRP1) and NLRP3 at 4 days or 8 weeks after radiation injury (P > 0.05). Furthermore, the expression levels of the inflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 were not significantly different among the groups (P > 0.05). The expression levels of cleaved caspase-1 and -3, indicators of apoptosis, were higher in the irradiation groups than in the control group at 4 days post irradiation, especially for caspase-3 (P < 0.05), but this increase was slightly attenuated at 8 weeks after radiation injury. Four days post irradiation, the MEMRI signal intensity (SI) in the irradiation groups, especially the 25 Gy group, was significantly lower than that in the control group (P < 0.05). Eight weeks after radiation injury, the SI of the 15 Gy group and the 25 Gy group recovered by different degrees, but the SI of the 25 Gy group was still significantly lower than that of the control group (P < 0.05). On day 4 post irradiation, the metabolic ratio of N-acetylaspartate (NAA) to creatine (Cr) in the 15 Gy group and 25 Gy group was significantly lower than that in the control group (P < 0.05). The NAA/Cr ratio in the 15 Gy group recovered to control levels at 8 weeks (P > 0.05), but the NAA/Cr ratio in the 25 Gy group remained significantly lower than that in the control group (P < 0.05). CONCLUSION: Radiation-induced brain injury is dose-dependently associated with apoptosis but not inflammasomes or pyroptosis, and the change in apoptosis can be detected by MEMRI.

Only IL-1ß release is inflammasome-dependent upon ultraviolet B irradiation although IL-18 is also secreted.

DNA damage accumulates in aged postmitotic retinal pigment epithelium (RPE) cells, a phenomenon associated with the development of age-related macular degeneration. In this study, we have experimentally induced DNA damage by ultraviolet B (UVB) irradiation in interleukin-1α (IL-1α)-primed ARPE-19 cells and examined inflammasome-mediated signaling. To reveal the mechanisms of inflammasome activation, cells were additionally exposed to high levels of extracellular potassium chloride, n-acetyl-cysteine, or mitochondria-targeted antioxidant MitoTEMPO, prior to UVB irradiation. Levels of interleukin-18 (IL-18) and IL-1ß mRNAs were detected with qRT-PCR and secreted amounts of IL-1ß, IL-18, and caspase-1 were measured with ELISA. The role of nucleotide-binding domain and leucine-rich repeat pyrin containing protein 3 (NLRP3) in UVB-induced inflammasome activation was verified by using the NLRP3-specific siRNA. Reactive oxygen species (ROS) levels were measured immediately after UVB exposure using the cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H2 DCFDA) indicator, the levels of cyclobutane pyrimidine dimers were assayed by cell-based ELISA, and the extracellular levels of adenosine triphosphate (ATP) determined using a commercial bioluminescence assay. We found that pro-IL-18 was constitutively expressed by ARPE-19 cells, whereas the expression of pro-IL-1ß was inducible by IL-1α priming. UVB induced the release of mature IL-18 and IL-1ß but NLRP3 contributed only to the secretion of IL-1ß. At the mechanistic level, the release of IL-1ß was regulated by K+ efflux, whereas the secretion of IL-18 was dependent on ROS production. As well as K+ efflux, the cells released ATP following UVB exposure. Collectively, our data suggest that UVB clearly stimulates the secretion of mature IL-18 as a result of ROS induction, and this response is associated with DNA damage. Moreover, in human RPE cells, K+ efflux mediates the UVB-activated NLRP3 inflammasome signaling, leading to the processing of IL-1ß.

Photo-Oxidative Blue-Light Stimulation in Retinal Pigment Epithelium Cells Promotes Exosome Secretion and Increases the Activity of the NLRP3 Inflammasome.

PURPOSE: Age-related macular degeneration (AMD) is a major cause of blindness in the elderly, and the activation of the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome is involved in AMD pathogenesis. We investigated whether photooxidative blue-light stimulation in retinal pigment epithelium (RPE) cells promotes exosome secretion and modulates the activity of the NLRP3 inflammasome in vitro. METHODS: Exosomes were isolated from ARPE-19 cultures stimulated or not with blue-light photostimulation (488 nm). Isolated exosomes were characterized by transmission electron microscope and Western blot analyses. The contents of the NLRP3 inflammasome (IL-1ß, IL-18, and caspase-1 as markers of the inflammasome) in exosomes were analyzed by Western blotting. After culture, IL-1ß, IL-18, and caspase-1 in RPE cells were analyzed by both immunofluorescence and Western blotting. RT-PCR and Western blotting were conducted to assess the contents of NLRP3 in RPE cells. RESULTS: Exosomes exhibited a typical characteristic morphology (cup-shaped) and size (diameter between 50 and 150 nm) in both groups. The exosome markers CD9, CD63, and CD81 were strongly present. After blue-light photostimulation, ARPE-19 cells were noted to release exosomes with higher levels of IL-1ß, IL-18, and caspase-1 than those in the control group. The levels of IL-1ß, IL-18, and caspase-1 in ARPE-19 cells were significantly enhanced when treated with stressed RPE exosomes. Additionally, the NLRP3 mRNA and protein levels were found to be markedly higher in the treated group than in the control group. CONCLUSIONS: Under photooxidative blue-light stimulation, RPE-derived exosomes may aggravate a potentially harmful oxidative response through the upregulation of the NLRP3 inflammasome.

Protective Effects of Flagellin A N/C Against Radiation-Induced NLR Pyrin Domain Containing 3 Inflammasome-Dependent Pyroptosis in Intestinal Cells.

PURPOSE: To investigate changes induced in mouse intestines after irradiation and to explore the potential radioprotective effects of flagellin A N/C (FlaAN/C). METHODS AND MATERIALS: A mouse model of radiation-induced enteropathy was used in this study. A 10-Gy abdominal irradiation was performed on FlaAN/C- and vehicle-injected mice to explore the role of FlaAN/C in intestinal radiation injury and to study the molecular mechanism in this process. In the intestinal tissue, pathologic changes were investigated by hematoxylin and eosin staining, TUNEL staining, and immunohistochemistry; Western blotting and quantitative reverse transcription-polymerase chain reaction were used to determine the changes in protein and messenger RNA levels, respectively; and an enzyme-linked immunosorbent assay was performed to determine protein concentration in serum. The involvement of the reactive oxygen species pathway was investigated by determining superoxide dismutase, glutathione (GSH) peroxidase, GSH reductase, and GSH disulfide (GSSG) plus GSH activities. RESULTS: Flagellin A N/C inhibited radiation-induced reactive oxygen species production, decreased NLRP3 activity, and reduced the occurrence of caspase-1-dependent pyroptosis. The results revealed that oxidative stress, bioenergetic impairment, and subsequent NLRP3 inflammasome activation were involved in radiation-induced intestinal injury. Flagellin A N/C exerted a protective effect by blunting the activation of NLRP3 inflammasome signaling, thereby reducing the inflammatory response and occurrence of caspase-1-dependent pyroptosis in the intestine. Therefore, treatment of mice with FlaAN/C reduced radiation-induced intestinal injury. CONCLUSIONS: Reactive oxygen species-induced NLRP3 inflammasomes mediated radiation-induced pyroptosis of the intestinal cells, and FlaAN/C suppressed pyroptosis to protect the intestinal tissue. These results provide novel insights into the mechanisms underlying radiation-induced cytotoxicity, and FlaAN/C might be a potential preventive therapy for radiation-induced intestinal injury in patients with cancer.

The DNA-sensing AIM2 inflammasome controls radiation-induced cell death and tissue injury.

Acute exposure to ionizing radiation induces massive cell death and severe damage to tissues containing actively proliferating cells, including bone marrow and the gastrointestinal tract. However, the cellular and molecular mechanisms underlying this pathology remain controversial. Here, we show that mice deficient in the double-stranded DNA sensor AIM2 are protected from both subtotal body irradiation-induced gastrointestinal syndrome and total body irradiation-induced hematopoietic failure. AIM2 mediates the caspase-1-dependent death of intestinal epithelial cells and bone marrow cells in response to double-strand DNA breaks caused by ionizing radiation and chemotherapeutic agents. Mechanistically, we found that AIM2 senses radiation-induced DNA damage in the nucleus to mediate inflammasome activation and cell death. Our results suggest that AIM2 may be a new therapeutic target for ionizing radiation exposure.

The Influence of Radiotherapy on AIM2 Inflammasome in Radiation Pneumonitis.

This study aims to investigate the influence of radiotherapy on absent in melanoma 2 (AIM2) inflammasome in radiation pneumonitis (RP). A rat model of RP was established. H&E staining was used to test radiation-induced lung tissue injury. Immunohistochemistry (IHC) was used to detect the expression of AIM2 and IL-1ß in rat lung tissues. Milliplex assay was used to test cytokine levels in rat serum. Comet assay was adopted to examine DNA breaks in THP1 cells. RT-PCR was used to detect the messenger RNA (mRNA) expression of AIM2, caspase-1, and IL-1ß in THP1 cells. As a result, the rat model indicated that irradiation induced obvious lung injury. A large amount of inflammatory cells infiltrated to the irradiated lung tissues. The structure of lung tissues collapsed. IHC revealed that AIM2 and IL-1ß expressions were significantly higher in irradiated lung tissues than in the control. IL-1ß level in rat serum significantly elevated on the 7th day post-irradiation, gradually decreased on the 15th day, and became minimal on the 30th day. Irradiation induced dsDNA break in a dose-dependent manner at 24 h after irradiation. Radiotherapy increased the mRNA expression level of AIM2 and IL-1ß in a time-dependent manner. In conclusion, radiotherapy triggered some critical components of AIM2 inflammasome in RP. The activation of AIM2 inflammasome by radiotherapy may contribute to the pathogenesis of RP.

Far-infrared promotes burn wound healing by suppressing NLRP3 inflammasome caused by enhanced autophagy.

UNLABELLED: Understanding the underlying molecular mechanisms in burn wound progression is crucial to providing appropriate diagnoses and designing therapeutic regimens for burn patients. When inflammation becomes unregulated, recurrent, or excessive, it interferes with burn wound healing. Autophagy, which is a homeostatic and catabolic degradation process, was found to protect against ischemic injury, inflammatory diseases, and apoptosis in some cases. In the present study, we investigated whether far-infrared (FIR) could ameliorate burn wound progression and promote wound healing both in vitro and in a rat model of deep second-degree burn. We found that FIR induced autophagy in differentiated THP-1 cells (human monocytic cells differentiated to macrophages). Furthermore, FIR inhibited both the NLRP3 inflammasome and the production of IL-1ß in lipopolysaccharide-activated THP-1 macrophages. In addition, FIR induced the ubiquitination of ASC, which is the adaptor protein of the inflammasome, by increasing tumor necrosis factor receptor-associated factor 6 (TRAF6), which is a ubiquitin E3 ligase. Furthermore, the exposure to FIR then promoted the delivery of inflammasome to autophagosomes for degradation. In a rat burn model, FIR ameliorated burn-induced epidermal thickening, inflammatory cell infiltration, and loss of distinct collagen fibers. Moreover, FIR enhanced autophagy and suppressed the activity of the NLRP3 inflammasome in the rat skin tissue of the burn model. Based on these results, we suggest that FIR-regulated autophagy and inflammasomes will be important for the discovery of novel therapeutics to promote the healing of burn wounds. KEY MESSAGES: Far-infrared (FIR) induced autophagy in THP-1 macrophages. FIR suppressed the NLRP3 inflammasome through the activation of autophagy. FIR induced the ubiquitination of ASC by increasing TRAF6. FIR ameliorated burn wound progression and promoted wound healing in a rat burn model.

Extracorporeal photopheresis promotes IL-1ß production.

Extracorporeal photopheresis (ECP) is a widely used clinical cell-based therapy exhibiting efficacy in heterogenous immune-mediated diseases such as cutaneous T cell lymphoma, graft-versus-host disease, and organ allograft rejection. Despite its documented efficacy in cancer immunotherapy, little is known regarding the induction of immunostimulatory mediators by ECP. In this article, we show that ECP promotes marked release of the prototypic immunostimulatory cytokine IL-1ß. ECP primes IL-1ß production and activates IL-1ß maturation and release in the context of caspase-1 activation in monocytes and myeloid dendritic cells. Of interest, IL-1ß maturation by ECP was fully intact in murine cells deficient in caspase-1, suggesting the predominance of an inflammasome-independent pathway for ECP-dependent IL-1ß maturation. Clinically, patient analysis revealed significantly increased IL-1ß production in stimulated leukapheresis concentrates and peripheral blood samples after ECP. Collectively, these results provide evidence for promotion of IL-1ß production by ECP and offer new insight into the immunostimulatory capacity of ECP.

Cathelicidin peptide LL-37 increases UVB-triggered inflammasome activation: possible implications for rosacea.

BACKGROUND: In patients with rosacea, environmental stressors, especially UVB radiation, trigger disease flares that are characterized by inflammation and vascular hyperactivity. An altered innate immune detection and response system, modulated to a large extent by the aberrant production and processing of human cathelicidin LL-37, is thought to play a central role in disease pathogenesis. OBJECTIVE: To investigate whether the proinflammatory and proangiogenic effects of UV radiation are enhanced in the presence of cathelicidin LL-37. METHODS: Human skin ex vivo and epidermal keratinocytes in vitro were exposed to UVB irradiation. The proinflammatory effects of UVB exposure in the presence and absence of LL-37 were characterized using immunoblot, transfection, qPCR, and a cell-based second messenger assay. ELISA was used to assess cytokine release and the angiogenic potential of endothelial cells was evaluated using an in vitro angiogenesis assay. RESULTS: UVB irradiation triggered the inflammasome-mediated processing and release of IL-1ß. LL-37 augmented this UV-induced IL-1ß secretion by acting on the P2X7 receptor on keratinocytes. P2X7 receptor activation by UVB and LL-37 resulted in an increase in intracellular calcium concentrations, which enhances inflammasome activation and subsequent IL-1ß release. Furthermore, IL-1ß and LL-37 worked synergistically to increase the angiogenic potential of endothelial cells. CONCLUSION: Cathelicidin LL-37 modulates the proinflammatory and proangiogenic effects of UV radiation and thereby contributes to enhanced sensitivity to sun exposure in rosacea.

Inflammasome activation of IL-1 family mediators in response to cutaneous photodamage.

Although keratinocytes are relatively resistant to ultraviolet radiation (UVR) induced damage, repeated UVR exposure result in accumulated DNA mutations that can lead to epidermal malignancies. Keratinocytes play a central role in elaborating innate responses that lead to inflammation and influence the generation of adaptive immune responses in skin. Apart from the minor cellular constituents of the epidermis, specifically Langerhans cells and melanocytes, keratinocytes are the major source of cytokines. UVR exposure stimulates keratinocytes to secrete abundant pro-inflammatory IL-1-family proteins, IL-1α, IL-1ß, IL-18, and IL-33. Normal skin contains only low levels of inactive precursor forms of IL-1ß and IL-18, which require caspase 1-mediated proteolysis for their maturation and secretion. However, caspase-1 activation is not constitutive, but dependents on the UV-induced formation of an active inflammasome complex. IL-1 family cytokines can induce a secondary cascade of mediators and cytokines from keratinocytes and other cells resulting in wide range of innate processes including infiltration of inflammatory leukocytes, induction of immunosuppression, DNA repair or apoptosis. Thus, the ability of keratinocytes to produce a wide repertoire of proinflammatory cytokines can influence the immune response locally as well as systematically, and alter the host response to photodamaged cells. We will highlight differential roles played by each IL-1 family molecule generated by UV-damaged keratinocytes, and reveal their complementary influences in modulating acute inflammatory and immunological events that follow cutaneous UV exposure.