IL-38 regulates intestinal stem cell homeostasis by inducing WNT signaling and beneficial IL-1ß secretion.

The IL-1 Family member IL-38 has been characterized primarily as an antiinflammatory cytokine in human and mouse models of systemic diseases. Here, we examined the role of IL-38 in the murine small intestine (SI). Immunostaining of SI revealed that IL-38 expression partially confines to intestinal stem cells. Cultures of intestinal organoids reveal IL-38 functions as a growth factor by increasing organoid size via inducing WNT3a. In contrast, organoids from IL-38-deficient mice develop more slowly. This reduction in size is likely due to the downregulation of intestinal stemness markers (i.e., Fzd5, Ephb2, and Olfm4) expression compared with wild-type organoids. The IL-38 binding to IL-1R6 and IL-1R9 is still a matter of debate. Therefore, to analyze the molecular mechanisms of IL-38 signaling, we also examined organoids from IL-1R9-deficient mice. Unexpectedly, these organoids, although significantly smaller than wild type, respond to IL-38, suggesting that IL-1R9 is not involved in IL-38 signaling in the stem cell crypt. Nevertheless, silencing of IL-1R6 disabled the organoid response to the growth property of IL-38, thus suggesting IL-1R6 as the main receptor used by IL-38 in the crypt compartment. In organoids from wild-type mice, IL-38 stimulation induced low concentrations of IL-1ß which contribute to organoid growth. However, high concentrations of IL-1ß have detrimental effects on the cultures that were prevented by treatment with recombinant IL-38. Overall, our data demonstrate an important regulatory function of IL-38 as a growth factor, and as an antiinflammatory molecule in the SI, maintaining homeostasis.

Recombinant IL-37 Exerts an Anti-inflammatory Effect on Human Aortic Valve Interstitial Cells through Extracellular and Intracellular Actions.

Calcific aortic valve disease (CAVD) is a chronic inflammatory disease with slow progression that involves soluble extracellular matrix (ECM) proteins. Previously, we found that recombinant interleukin (IL)-37 suppresses aortic valve interstitial cells (AVIC) inflammatory response through the interaction with IL-18 receptor α-chain (IL-18Rα) on the cell surface. Endogenous IL-37 can be retained in the cytoplasm or released into extracellular spaces. It remains unknown whether recombinant IL-37 exerts the anti-inflammatory effect through intracellular action. Here, we found that recombinant IL-37 suppressed AVIC inflammatory response to soluble ECM proteins. Interestingly, recombinant IL-37 was internalized by human AVICs in an IL-18Rα-independent fashion. Blocking endocytic pathways reduced the internalization and anti-inflammatory potency of recombinant IL-37. Overexpression of IL-37 in human AVICs suppressed soluble ECM proteins-induced NF-κB activation and the production of ICAM-1 and VCAM-1. However, IL-37D20A (mutant IL-37 lacking nucleus-targeting sequences) overexpression had no such effect, and the inflammatory response to soluble ECM proteins was essentially intact in AVICs from transgenic mice expressing IL-37D20A. Together, recombinant IL-37 can be internalized by human AVICs through endocytosis. Intracellular IL-37 exerts an anti-inflammatory effect through a nucleus-targeting mechanism. This study highlights the potent anti-inflammatory effect of recombinant IL-37 in both extracellular and intracellular compartments through distinct mechanisms.

Oligomer-to-monomer transition underlies the chaperone function of AAGAB in AP1/AP2 assembly.

Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin-binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homodimer. AAGAB dimerization is mediated by its C-terminal domain (CTD), which is critical for AAGAB stability and is missing in mutant proteins found in patients with the skin disease punctate palmoplantar keratoderma type 1 (PPKP1). We solved the crystal structure of the dimerization-mediating CTD, revealing an antiparallel dimer of bent helices. Interestingly, AAGAB uses the same CTD to recognize and stabilize the γ subunit in the AP1 complex and the α subunit in the AP2 complex, forming binary complexes containing only one copy of AAGAB. These findings demonstrate a dual role of CTD in stabilizing resting AAGAB and binding to substrates, providing a molecular explanation for disease-causing AAGAB mutations. The oligomerization state transition mechanism may also underlie the functions of other assembly chaperones.

Activation of Host-NLRP3 Inflammasome in Myeloid Cells Dictates Response to Anti-PD-1 Therapy in Metastatic Breast Cancers.

Tumor-associated inflammation leads to dysregulated cytokine production that promotes tumor immune evasion and anti-tumor immunity dysfunction. In advanced stage breast cancer, the proinflammatory cytokine IL-1ß is overexpressed due to large proportions of activated myeloid cells in the tumor microenvironment (TME). Here, we demonstrate the role of the host nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing 3 (NLRP3) inflammasome in metastatic breast cancer. In vitro, we show that stimulation of THP-1 cells with conditioned media collected from MDA-MB-468 cells induced NLRP3 activation and increased Pdcd1l1 expression. In vivo, mice deficient in NLRP3 orthotopically implanted with metastatic breast cancer cell line (E0771) showed significant reduction in tumor growth (p < 0.05) and increased survival (p < 0.01). Inhibition of NLRP3 with the small molecule OLT1177® reduced expression of Pdcd1l1 (p < 0.001), Casp1 (p < 0.01) and Il1b (p < 0.01) in primary tumors. Furthermore, tumor-bearing mice receiving OLT1177® showed reduced infiltration of myeloid-derived suppressor cells (MDSCs) (p < 0.001) and increased CD8+ T cells (p < 0.05) and NK cells (p < 0.05) in the TME. NLRP3 inhibition in addition to anti-PD-1 treatment significantly reduced tumor growth from the monotherapies (p < 0.05). These data define NLRP3 activation as a key driver of immune suppression in metastatic breast cancers. Furthermore, this study suggests NLRP3 as a valid target to increase efficacy of immunotherapy with checkpoint inhibitor in metastatic breast cancers.

AAGAB is an assembly chaperone regulating AP1 and AP2 clathrin adaptors.

Multimeric cargo adaptors such as AP2 play central roles in intracellular membrane trafficking. We recently discovered that the assembly of the AP2 adaptor complex, a key player in clathrin-mediated endocytosis, is a highly organized process controlled by alpha- and gamma-adaptin-binding protein (AAGAB, also known as p34). In this study, we demonstrate that besides AP2, AAGAB also regulates the assembly of AP1, a cargo adaptor involved in clathrin-mediated transport between the trans-Golgi network and the endosome. However, AAGAB is not involved in the formation of other adaptor complexes, including AP3. AAGAB promotes AP1 assembly by binding and stabilizing the γ and σ subunits of AP1, and its mutation abolishes AP1 assembly and disrupts AP1-mediated cargo trafficking. Comparative proteomic analyses indicate that AAGAB mutation massively alters surface protein homeostasis, and its loss-of-function phenotypes reflect the synergistic effects of AP1 and AP2 deficiency. Taken together, these findings establish AAGAB as an assembly chaperone for both AP1 and AP2 adaptors and pave the way for understanding the pathogenesis of AAGAB-linked diseases.

A novel anti-human IL-1R7 antibody reduces IL-18-mediated inflammatory signaling.

Unchecked inflammation can result in severe diseases with high mortality, such as macrophage activation syndrome (MAS). MAS and associated cytokine storms have been observed in COVID-19 patients exhibiting systemic hyperinflammation. Interleukin-18 (IL-18), a proinflammatory cytokine belonging to the IL-1 family, is elevated in both MAS and COVID-19 patients, and its level is known to correlate with the severity of COVID-19 symptoms. IL-18 binds its specific receptor IL-1 receptor 5 (IL-1R5, also known as IL-18 receptor alpha chain), leading to the recruitment of the coreceptor, IL-1 receptor 7 (IL-1R7, also known as IL-18 receptor beta chain). This heterotrimeric complex then initiates downstream signaling, resulting in systemic and local inflammation. Here, we developed a novel humanized monoclonal anti-IL-1R7 antibody to specifically block the activity of IL-18 and its inflammatory signaling. We characterized the function of this antibody in human cell lines, in freshly obtained peripheral blood mononuclear cells (PBMCs) and in human whole blood cultures. We found that the anti-IL-1R7 antibody significantly suppressed IL-18-mediated NFκB activation, reduced IL-18-stimulated IFNγ and IL-6 production in human cell lines, and reduced IL-18-induced IFNγ, IL-6, and TNFα production in PBMCs. Moreover, the anti-IL-1R7 antibody significantly inhibited LPS- and Candida albicans-induced IFNγ production in PBMCs, as well as LPS-induced IFNγ production in whole blood cultures. Our data suggest that blocking IL-1R7 could represent a potential therapeutic strategy to specifically modulate IL-18 signaling and may warrant further investigation into its clinical potential for treating IL-18-mediated diseases, including MAS and COVID-19.

Extracellular and nuclear roles of IL-37 after spinal cord injury.

Interleukin 37 (IL-37) is an anti-inflammatory cytokine of the interleukin 1 family. Transgenic mice expressing the human form of the IL37 gene (hIL-37Tg) display protective effects in several animal models of disease. Previous data from our group revealed that IL-37 limits inflammation after spinal cord injury (SCI) and ameliorates tissue damage and functional deficits. IL-37 can exert its anti-inflammatory effects by translocating to the nucleus or acting as an extracellular cytokine. However, whether this protection after SCI is mediated by translocating to the nucleus, activating of extracellular receptors, or both, is currently unknown. In the present study, we used different transgenic animals to answer this question. We demonstrated that the beneficial effects of IL-37 on functional and histological outcomes after SCI were lost in the lack of the extracellular receptor IL-1R8, indicating that IL-37 induces protection as an extracellular cytokine. On the other hand, transgenic mice with the nuclear function of IL-37 abolished (hIL-37D20ATg) showed significant improvement in locomotor skills and myelin sparing after SCI, indicating that nuclear pathway is not required for the protective actions of IL-37. Moreover, we also showed that the therapeutic effects of the recombinant IL-37 protein are produced only in the presence of the extracellular receptor IL-1R8, further highlighting the importance of the extracellular function of this cytokine after SCI. Finally, we revealed that the administration of recombinant IL-37 protein exerted therapeutic actions when administered in the lesion site but not systemically. This work demonstrated for the first time that translocation of IL-37 to the nucleus is not required for the beneficial actions of this cytokine after SCI and highlights the importance of the extracellular signaling of IL-37 to mediate neuroprotective actions.

Human recombinant interleukin-38 suppresses inflammation in mouse models of local and systemic disease.

Interleukin (IL)-38 belongs to the IL-1 family and is part of the IL-36 subfamily due to its binding to the IL-36 Receptor (IL-1R6). In the current study, we assessed the anti-inflammatory properties of IL-38 in murine models of arthritis and systemic inflammation. First, the anti-inflammatory properties of mouse and human IL-38 precursors were compared to forms with a truncated N-terminus. In mouse bone marrow derived dendritic cells (BMDC), human and mouse IL-38 precursors with a truncation of the two N-terminal amino acids (3-152) suppressed LPS-induced IL-6. Recombinant human IL-38 (3-152) was further investigated for its immunomodulatory potential using four murine models of inflammatory disease: streptococcal cell wall (SCW)-induced arthritis, monosodium urate (MSU) crystal-induced arthritis, MSU crystal-induced peritonitis, and systemic endotoxemia. In each of these models IL-38 significantly reduced inflammation. In SCW and MSU crystal-induced arthritis, joint swelling, inflammatory cell influx, and synovial levels of IL-1ß, IL-6, and KC were reduced by 50% or greater. These suppressive properties of IL-38 in SCW-induced arthritis were independent of the anti-inflammatory co-receptor IL-1R8, as IL-38 reduced arthritis equally in IL-1R8 deficient and WT mice. In MSU crystal-induced peritonitis, IL-38 reduced hypothermia, while plasma IL-6 and KC and peritoneal KC levels were reduced by 65-70%. In the LPS endotoxemia model, IL-38 pretreatment reduced systemic IL-6, TNFα and KC. Furthermore, in ex vivo cultured bone marrow, LPS-induced IL-6, TNFα and KC were reduced by 75-90%. Overall, IL-38 exhibits broad anti-inflammatory properties in models of systemic and local inflammation and therefore may be an effective cytokine therapy.

PBRM1 and the glycosylphosphatidylinositol biosynthetic pathway promote tumor killing mediated by MHC-unrestricted cytotoxic lymphocytes.

Major histocompatibility complex (MHC)-unrestricted cytotoxic lymphocytes (CLs) such as natural killer (NK) cells can detect and destroy tumor and virus-infected cells resistant to T cell-mediated killing. Here, we performed genome-wide genetic screens to identify tumor-intrinsic genes regulating killing by MHC-unrestricted CLs. A group of genes identified in our screens encode enzymes for the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor, which is not involved in tumor response to T cell-mediated cytotoxicity. Another gene identified in the screens was PBRM1, which encodes a subunit of the PBAF form of the SWI/SNF chromatin-remodeling complex. PBRM1 mutations in tumor cells cause resistance to MHC-unrestricted killing, in contrast to their sensitizing effects on T cell-mediated killing. PBRM1 and the GPI biosynthetic pathway regulate the ligands of NK cell receptors in tumor cells and promote cytolytic granule secretion in CLs. The regulators identified in this work represent potential targets for cancer immunotherapy.

Role for nuclear interleukin-37 in the suppression of innate immunity.

The IL-1 family member IL-37 broadly suppresses innate inflammation and acquired immunity. Similar to IL-1α and IL-33, IL-37 is a dual-function cytokine in that IL-37 translocates to the nucleus but also transmits a signal via surface membrane receptors. The role of nuclear IL-37 remains unknown on the ability of this cytokine to inhibit innate inflammation. Here, we compared suppression of innate inflammation in transgenic mice expressing native human IL-37 (IL-37Tg) with those of transgenic mice carrying the mutation of aspartic acid (D) to alanine (A) at amino acid 20 (IL-37D20ATg). The mutation D20A prevents cleavage of caspase-1, a step required for IL-37 nuclear translocation. In vitro, peritoneal macrophages from IL-37Tg mice reduced LPS-induced IL-1ß, IL-6, TNFα and IFNγ by 40-50% whereas in macrophages from IL-37D20ATg mice this suppression was not observed, consistent with loss of nuclear function. Compared with macrophages from IL-37Tg mice, significantly less or no suppression of LPS-induced MAP kinase and NFκB activation was also observed in macrophages from IL-37D20ATg mice. In vivo, levels of IL-1ß, IL-6, and TNFα in the lungs and liver were markedly reduced during endotoxemia in IL-37Tg mice but not observed in IL-37D20ATg mice. However, suppression of innate inflammation remains intact in the IL-37D20A mice once the cytokine is released from the cell and binds to its receptor. These studies reveal a nuclear function for suppression of innate inflammation and are consistent with the dual function of IL-37 and a role for caspase-1 in limiting inflammation.

The selective ROCK2 inhibitor KD025 reduces IL-17 secretion in human peripheral blood mononuclear cells independent of IL-1 and IL-6.

Reducing the activities of the pro-inflammatory cytokine IL-17 is an effective treatment strategy for several chronic autoimmune disorders. Rho-associated coiled-coil containing kinase 2 (ROCK2) is a member of the serine-threonine protein kinase family that regulates IL-17 secretion in T cells via signal transducer and activator of transcription 3 (STAT3)-dependent mechanism. We reported here that the selective ROCK2 inhibitor KD025 significantly reduced in vitro production of IL-17 in unfractionated human peripheral blood mononuclear cells (PBMCs) stimulated with the dectin-1 agonist Candida albicans. C. albicans induced IL-17 was reduced by 70% (p < 0.0001); a similar reduction (80%) was observed in PBMC stimulated with the Toll-like receptor 2 agonist Staphylococcus epidermidis (p < 0.0001). Treatment of PBMC with KD025 was not associated with a reduction in IL-1ß, IL-6 or IL-1α levels; in contrast, a 1.5 fold increase in the level of IL-1 receptor antagonist (IL-1Ra) was observed (p < 0.001). KD025 down-regulated C. albicans-induced Myosin Light Chain and STAT3, whereas STAT5 phosphorylation increased. Using anti-CD3/CD28 activation of the TCR, KD025 similarly suppressed IL-17 independent of a reduction in IL-1ß. Thus, ROCK2 directly regulates IL-17 secretion independent of endogenous IL-1 and IL-6 supporting development of selective ROCK2 inhibitors for treatment of IL-17-driven inflammatory diseases.

Interleukin-37 treatment of mice with metabolic syndrome improves insulin sensitivity and reduces pro-inflammatory cytokine production in adipose tissue.

Obesity and the metabolic syndrome are characterized by chronic, low-grade inflammation mainly originating from expanding adipose tissue and resulting in inhibition of insulin signaling and disruption of glycemic control. Transgenic mice expressing human interleukin 37 (IL-37), an anti-inflammatory cytokine of the IL-1 family, are protected against metabolic syndrome when fed a high-fat diet (HFD) containing 45% fat. Here, we examined whether treatment with recombinant IL-37 ameliorates established insulin resistance and obesity-induced inflammation. WT mice were fed a HFD for 22 weeks and then treated daily with IL-37 (1 µg/mouse) during the last 2 weeks. Compared with vehicle only-treated mice, IL-37-treated mice exhibited reduced insulin in the plasma and had significant improvements in glucose tolerance and in insulin content of the islets. The IL-37 treatment also increased the levels of circulating IL-1 receptor antagonist. Cultured adipose tissues revealed that IL-37 treatment significantly decreases spontaneous secretions of IL-1ß, tumor necrosis factor α (TNFα), and CXC motif chemokine ligand 1 (CXCL-1). We also fed mice a 60% fat diet with concomitant daily IL-37 for 2 weeks and observed decreased secretion of IL-1ß, TNFα, and IL-6 and reduced intracellular levels of IL-1α in the liver and adipose tissue, along with improved plasma glucose clearance. Compared with vehicle treatment, these IL-37-treated mice had no apparent weight gain. In human adipose tissue cultures, the presence of 50 pm IL-37 reduced spontaneous release of TNFα and 50% of lipopolysaccharide-induced TNFα. These findings indicate that IL-37's anti-inflammatory effects can ameliorate established metabolic disturbances during obesity.

Interleukin-37 suppresses the osteogenic responses of human aortic valve interstitial cells in vitro and alleviates valve lesions in mice.

Calcific aortic valve disease is a chronic inflammatory process, and aortic valve interstitial cells (AVICs) from diseased aortic valves express greater levels of osteogenic factors in response to proinflammatory stimulation. Here, we report that lower cellular levels of IL-37 in AVICs of diseased human aortic valves likely account for augmented expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphatase (ALP) following stimulation of Toll-like receptor (TLR) 2 or 4. Treatment of diseased AVICs with recombinant human IL-37 suppresses the levels of BMP-2 and ALP as well as calcium deposit formation. In mice, aortic valve thickening is observed when exposed to a TLR4 agonist or a high fat diet for a prolonged period; however, mice expressing human IL-37 exhibit significantly lower BMP-2 levels and less aortic valve thickening when subjected to the same regimens. A high fat diet in mice results in oxidized low-density lipoprotein (oxLDL) deposition in aortic valve leaflets. Moreover, the osteogenic responses in human AVICs induced by oxLDL are suppressed by recombinant IL-37. Mechanistically, reduced osteogenic responses to oxLDL in human AVICs are associated with the ability of IL-37 to inhibit NF-κB and ERK1/2. These findings suggest that augmented expression of osteogenic factors in AVICs of diseased aortic valves from humans is at least partly due to a relative IL-37 deficiency. Because recombinant IL-37 suppresses the osteogenic responses in human AVICs and alleviates aortic valve lesions in mice exposed to high fat diet or a proinflammatory stimulus, IL-37 has therapeutic potential for progressive calcific aortic valve disease.

Suppression of innate inflammation and immunity by interleukin-37.

IL-37 is unique in the IL-1 family in that unlike other members of the family, IL-37 broadly suppresses innate immunity. IL-37 can be elevated in humans with inflammatory and autoimmune diseases where it likely functions to limit inflammation. Transgenic mice expressing human IL-37 (IL37-tg) exhibit less severe inflammation in models of endotoxin shock, colitis, myocardial infarction, lung, and spinal cord injury. IL37-tg mice have reduced antigen-specific responses and dendritic cells (DCs) from these mice exhibit characteristics of tolerogenic DCs. Compared to aging wild-type (WT) mice, aging IL37-tg mice are protected against B-cell leukemogenesis and heart failure. Treatment of WT mice with recombinant human IL-37 has been shown to be protective in several models of inflammation and injury. IL-37 binds to the IL-18 receptor but then recruits the orphan IL-1R8 (formerly TIR8 or SIGIRR) in order to function as an inhibitor. Here, we review the discovery of IL-37, its production, release, and mechanisms by which IL-37 reduces inflammation and suppresses immune responses. The data reviewed here suggest a therapeutic potential for IL-37.

IL-37 requires the receptors IL-18Rα and IL-1R8 (SIGIRR) to carry out its multifaceted anti-inflammatory program upon innate signal transduction.

Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37-IL-1R8-IL-18Rα. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Rα impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-κB, as well as mitogen-activated protein kinases. Furthermore, IL-37-IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Rα and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program.

Extracellular forms of IL-37 inhibit innate inflammation in vitro and in vivo but require the IL-1 family decoy receptor IL-1R8.

Similar to IL-1α and IL-33, IL-1 family member IL-37b translocates to the nucleus and is associated with suppression of innate and adaptive immunity. Here we demonstrate an extracellular function of the IL-37 precursor and a processed form. Recombinant IL-37 precursor reduced LPS-induced IL-6 by 50% (P < 0.001) in highly inflammatory human blood-derived M1 differentiated macrophages derived from selective subjects but not M2 macrophages. In contrast, a neutralizing monoclonal anti-IL-37 increased LPS-induced IL-6, TNFα and IL-1ß (P < 0.01). The suppression by IL-37 was consistently observed at low picomolar but not nanomolar concentrations. Whereas LPS induced a 12-fold increase in TNFα mRNA, IL-37 pretreatment decreased the expression to only 3-fold over background (P < 0.01). Mechanistically, LPS-induced p38 and pERK were reduced by IL-37. Recombinant IL-37 bound to the immobilized ligand binding α-chain of the IL-18 receptor as well as to the decoy receptor IL-1R8. In M1 macrophages, LPS increased the surface expression of IL-1R8. Compared with human blood monocytes, resting M1 cells express more surface IL-1R8 as well as total IL-1R8; there was a 16-fold increase in IL-1R8 mRNA levels when pretreated with IL-37. IL-37 reduced LPS-induced TNFα and IL-6 by 50-55% in mouse bone marrow-derived dendritic cells, but not in dendritic cells derived from IL-1R8-deficient mice. In mice subjected to systemic LPS-induced inflammation, pretreatment with IL-37 reduced circulating and organ cytokine levels. Thus, in addition to a nuclear function, IL-37 acts as an extracellular cytokine by binding to the IL-18 receptor but using the IL-1R8 for its anti-inflammatory properties.

Specific inhibition of histone deacetylase 8 reduces gene expression and production of proinflammatory cytokines in vitro and in vivo.

ITF2357 (generic givinostat) is an orally active, hydroxamic-containing histone deacetylase (HDAC) inhibitor with broad anti-inflammatory properties, which has been used to treat children with systemic juvenile idiopathic arthritis. ITF2357 inhibits both Class I and II HDACs and reduces caspase-1 activity in human peripheral blood mononuclear cells and the secretion of IL-1ß and other cytokines at 25-100 nm; at concentrations >200 nm, ITF2357 is toxic in vitro. ITF3056, an analog of ITF2357, inhibits only HDAC8 (IC50 of 285 nm). Here we compared the production of IL-1ß, IL-1α, TNFα, and IL-6 by ITF2357 with that of ITF3056 in peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), heat-killed Candida albicans, or anti-CD3/anti-CD28 antibodies. ITF3056 reduced LPS-induced cytokines from 100 to 1000 nm; at 1000 nm, the secretion of IL-1ß was reduced by 76%, secretion of TNFα was reduced by 88%, and secretion of IL-6 was reduced by 61%. The intracellular levels of IL-1α were 30% lower. There was no evidence of cell toxicity at ITF3056 concentrations of 100-1000 nm. Gene expression of TNFα was markedly reduced (80%), whereas IL-6 gene expression was 40% lower. Although anti-CD3/28 and Candida stimulation of IL-1ß and TNFα was modestly reduced, IFNγ production was 75% lower. Mechanistically, ITF3056 reduced the secretion of processed IL-1ß independent of inhibition of caspase-1 activity; however, synthesis of the IL-1ß precursor was reduced by 40% without significant decrease in IL-1ß mRNA levels. In mice, ITF3056 reduced LPS-induced serum TNFα by 85% and reduced IL-1ß by 88%. These data suggest that specific inhibition of HDAC8 results in reduced inflammation without cell toxicity.

IL-37 inhibits inflammasome activation and disease severity in murine aspergillosis.

Since IL-37 transgenic mice possesses broad anti-inflammatory properties, we assessed whether recombinant IL-37 affects inflammation in a murine model of invasive pulmonary aspergillosis. Recombinant human IL-37 was injected intraperitoneally into mice prior to infection and the effects on lung inflammation and inflammasome activation were evaluated. IL-37 markedly reduced NLRP3-dependent neutrophil recruitment and steady state mRNA levels of IL-1ß production and mitigated lung inflammation and damage in a relevant clinical model, namely aspergillosis in mice with cystic fibrosis. The anti-inflammatory activity of IL-37 requires the IL-1 family decoy receptor TIR-8/SIGIRR. Thus, by preventing activation of the NLRP3 inflammasome and reducing IL-1ß secretion, IL-37 functions as a broad spectrum inhibitor of the innate response to infection-mediated inflammation, and could be considered to be therapeutic in reducing the pulmonary damage due to non-resolving Aspergillus infection and disease.