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We recently described the induction of noncanonical IL-1β processing via caspase-8 recruited to ripoptosome signaling platforms in myeloid leukocytes. Here, we demonstrate that activated NLRP3·ASC inflammasomes recruit caspase-8 to drive IL-1β processing in murine bone marrow-derived dendritic cells (BMDC) independent of caspase-1 and -11. Sustained stimulation (2 h) of LPS-primed caspase-1-deficient (Casp1/11(-/-)) BMDC with the canonical NLRP3 inflammasome agonist nigericin results in release of bioactive IL-1β in conjunction with robust caspase-8 activation. This IL-1β processing and caspase-8 activation do not proceed in Nlrp3(-/-) or Asc(-/-) BMDC and are suppressed by pharmacological inhibition of caspase-8, indicating that caspase-8 can act as a direct IL-1β-converting enzyme during NLRP3 inflammasome activation. In contrast to the rapid caspase-1-mediated death of wild type (WT) BMDC via NLRP3-dependent pyroptosis, nigericin-stimulated Casp1/11(-/-) BMDC exhibit markedly delayed cell death via NLRP3-dependent apoptosis. Biochemical analyses of WT and Casp1/11(-/-) BMDC indicated that caspase-8 is proteolytically processed within detergent-insoluble ASC-enriched protein complexes prior to extracellular export during nigericin treatment. Although nigericin-stimulated caspase-1 activation and activity are only modestly attenuated in caspase-8-deficient (Casp8(-/-)Rip3(-/-)) BMDC, these cells do not exhibit the rapid loss of viability of WT cells. These results support a contribution of caspase-8 to both IL-1β production and regulated death signaling via NLRP3 inflammasomes. In the absence of caspase-1, NLRP3 inflammasomes directly utilize caspase-8 as both a pro-apoptotic initiator and major IL-1β-converting protease. In the presence of caspase-1, caspase-8 acts as a positive modulator of the NLRP3-dependent caspase-1 signaling cascades that drive both IL-1β production and pyroptotic death.

The identification of noncanonical (caspase-1–independent) pathways for IL-1β production has unveiled an intricate interplay between inflammatory and death-inducing signaling platforms. We found a heretofore unappreciated role for caspase-8 as a major pathway for IL-1β processing and release in murine bone marrow–derived dendritic cells (BMDC) costimulated with TLR4 agonists and proapoptotic chemotherapeutic agents such as doxorubicin (Dox) or staurosporine (STS). The ability of Dox to stimulate release of mature (17-kDa) IL-1β was nearly equivalent in wild-type (WT) BMDC, Casp1−/−Casp11−/− BMDC, WT BMDC treated with the caspase-1 inhibitor YVAD, and BMDC lacking the inflammasome regulators ASC, NLRP3, or NLRC4. Notably, Dox-induced production of mature IL-1β was temporally correlated with caspase-8 activation in WT cells and greatly suppressed in Casp8−/−Rip3−/− or Trif−/− BMDC, as well as in WT BMDC treated with the caspase-8 inhibitor, IETD. Similarly, STS stimulated robust IL-1β processing and release in Casp1−/−Casp11−/− BMDC that was IETD sensitive. These data suggest that TLR4 induces assembly of caspase-8–based signaling complexes that become licensed as IL-1β–converting enzymes in response to Dox and STS. The responses were temporally correlated with downregulation of cellular inhibitor of apoptosis protein 1, suggesting suppressive roles for this and likely other inhibitor of apoptosis proteins on the stability and/or proteolytic activity of the caspase-8 platforms. Thus, proapoptotic chemotherapeutic agents stimulate the caspase-8–mediated processing and release of IL-1β, implicating direct effects of such drugs on a noncanonical inflammatory cascade that may modulate immune responses in tumor microenvironments.

Purpose Previous reports have identified noninfectious uveitis as a potential sequela following both intravitreal bevacizumab and ranibizumab injections. We present two unique cases of acute anterior uveitis following intravitreal bevacizumab that did not occur with subsequent ranibizumab injections. Methods Case report. Conclusion These cases may reflect differences in the etiology of anterior uveitis following intravitreal bevacizumab and ranibizumab. Given these differences, it may be reasonable to offer ranibizumab to patients who have experienced presumed bevacizumab-induced anterior uveitis.

Interleukin-1β (IL-1β) can limit tumor growth by promoting T cell-mediated antitumor immune responses. Several chemotherapeutic agents can stimulate the production of IL-1β by tumor-infiltrating leukocytes via the NLRP3 inflammasome. We have recently demonstrated that some chemotherapeutics can also trigger the secretion of IL-1β by driving the assembly of the caspase-8- and FADD-containing platform known as the ripoptosome.

Diabetic macular edema represents one of the most important causes of visual morbidity in diabetes mellitus. The National Diabetes Information Clearinghouse estimates the prevalence of diabetes mellitus types 1 and 2 at 11.3% of the population above the age of 20, with an annual incidence of 1.9 million cases in the United States alone. In this population, the prevalence of diabetic macular edema is estimated at 30% of patients inflicted by the disease for 20 years or more. Diabetes mellitus is the leading cause of preventable blindness owing to both diabetic macular edema and complications of proliferative diabetic retinopathy (NDIC 2011). Thus once can expect that diabetic macular edema is a common entity in any Retina specialty practice with serious implications for vision loss if not treated in a timely and appropriate manner. Based on the critical findings of the Early Treatment in Diabetic Retinopathy Study, the standard of care has been focal laser photocoagulation therapy along with strong recommendations for strict blood glucose and blood pressure control. However since then, the spectrum of therapies for diabetic macular edema has expanded and continues to evolve. The use of steroid therapy and anti-vascular endothelial growth factor biologics have been compared to focal laser photocoagulation in order to establish more treatment options with equivalent efficacy and safety.

Human adenylosuccinate lyase (ASL) deficiency is an inherited metabolic disease in which the majority of the patients are compound heterozygotes for the mutations that occur in the ASL gene. Starting with purified wild-type (WT) and single-mutant human ASL, we generated in vitro hybrids that mimic compound heterozygote ASL. For this study, we used His-tagged WT/non-His-tagged WT, His-tagged WT/non-His-tagged R396C, His-tagged WT/non-His-tagged R396H, His-tagged R194C/non-His-tagged R396C, and His-tagged L311V/non-His-tagged R396H enzyme pairs. We generated various hybrids by denaturing pairs of enzymes in 1 M guanidinium chloride and renaturing them by removing the denaturant. The hybrids were separated on a nickel-nitrilotriacetic acid-agarose column based on the number of His tags present in the enzyme tetramer. Analytical ultracentrifuge data indicate that the hybrids have predominant amounts of heterotetramers. Analysis of the V(max) values of the hybrids indicates that most of the subunits behave independently; however, the hybrid tetramers retain weak positive cooperativity, indicating that there is some interaction between the different subunit types. The interactions between WT and mutant subunits may be advantageous to the parents of ASL deficient patients, while the interactions between some mutant subunits may assist heterozygote ASL deficient patients.

The emergence of non-canonical (caspase-1 independent) IL-1β production has unveiled an intricate interplay between inflammatory and death-inducing signaling platforms. We compared the relative contributions of canonical versus non-canonical IL-1β processing in murine bone marrow-derived dendritic cells (BMDC) stimulated with pro-apoptotic chemotherapeutic agents such as doxorubicin (Dox). Dox induced a release of mature (17 kDa) IL-1β from LPS-primed BMDC that was temporally correlated with caspase-8 activation and greatly suppressed in BMDC from Casp8-/-Rip3-/- mice. The magnitude of Dox-stimulated IL-1β release from Casp1-/-Casp11-/- BMDC or wild-type (WT) BMDC treated with YVAD caspase-1 inhibitor was similar to that in non-YVAD-treated WT BMDC. Neither ASC nor NLRP3 were required for Dox-stimulated IL-1β release. In contrast, Dox-induced IL-1β release was suppressed in LPS-primed TRIF-/- BMDC. This indicated interaction with a TLR4-TRIF pathway for assembly of RIP1/FADD/procaspase-8 ripoptosome complexes. Current studies focus on characterizing the ability of Dox to induce down-regulation of cIAPs and/or cFLIP which act to restrain the stability/proteolytic activity of caspase-8 containing ripoptosomes. These findings show that Dox promotes caspase-8-dependent release of IL-1β, implicating direct effects of such genotoxic chemotherapeutic drugs on a non-canonical inflammatory cascade that may modulate immune responses in tumor microenvironments.

The success of certain cancer chemotherapy drugs relies critically on the host immune system. In some cancer models, induction of NLRP3/caspase-1/IL-1β signaling cascades in dendritic cells is required for effective anti-tumor immunity. Recent studies by Sauter et al. (Cancer Biol Ther 2010) reported (and we have confirmed) that the chemotherapeutic anthracycline, doxorubicin (DOX), induces NLRP3 inflammasome-dependent IL-1β secretion from murine macrophages. We have also tested several other chemotherapy drugs for their ability to induce IL-1β secretion and/or apoptotic induction in LPS-primed murine bone marrow-derived macrophages (BMDM). Staurosporine (ST), a pro-apoptotic kinase inhibitor, was identified as another rapid inducer of caspase-1 activation and IL-1β release; ST initially (over the first 3 h) activates a K+ efflux-independent casp-1 activation but secondarily engages a K+ efflux-dependent pathway at later (>3h) treatment times. Both DOX and ST also induce rapid (within 1-4 h) accumulation of casp-3/7 activity which coincides with, or precedes, the casp-1 activation and IL-1β secretion responses. Ongoing studies are testing whether: 1) casp-3/7-mediated cleavage of the pannexin-1 channel contributes to the K+ efflux required for NLRP3 inflammasome activation by DOX; and 2) NLRP3-independent inflammasomes mediate the initial K+ efflux-independent IL-1β release elicited by ST.