Granzyme B is a cytotoxic lymphocyte-derived protease that plays a central role in promoting apoptosis of virus-infected target cells, through direct proteolysis and activation of constituents of the cell death machinery. However, previous studies have also implicated granzymes A and B in the production of proinflammatory cytokines, via a mechanism that remains undefined. Here we show that IL-1α is a substrate for granzyme B and that proteolysis potently enhanced the biological activity of this cytokine in vitro as well as in vivo. Consistent with this, compared with full-length IL-1α, granzyme B-processed IL-1α exhibited more potent activity as an immunoadjuvant in vivo. Furthermore, proteolysis of IL-1α within the same region, by proteases such as calpain and elastase, was also found to enhance its biological potency. Thus, IL-1α processing by multiple immune-related proteases, including granzyme B, acts as a switch to enhance the proinflammatory properties of this cytokine.
Apoptosis is commonly thought to represent an immunologically silent or even anti-inflammatory mode of cell death, resulting in cell clearance in the absence of explicit activation of the immune system. However, here we show that Fas/CD95-induced apoptosis is associated with the production of an array of cytokines and chemokines, including IL-6, IL-8, CXCL1, MCP-1, and GMCSF. Fas-induced production of MCP-1 and IL-8 promoted chemotaxis of phagocytes toward apoptotic cells, suggesting that these factors serve as "find-me" signals in this context. We also show that RIPK1 and IAPs are required for optimal production of cytokines and chemokines in response to Fas receptor stimulation. Consequently, a synthetic IAP antagonist potently suppressed Fas-dependent expression of multiple proinflammatory mediators and inhibited Fas-induced chemotaxis. Thus, in addition to provoking apoptosis, Fas receptor stimulation can trigger the secretion of chemotactic factors and other immunologically active proteins that can influence immune responsiveness toward dying cells.
Despite its long record of successful use in human vaccines, the mechanisms underlying the immunomodulatory effects of alum are not fully understood. Alum is a potent inducer of interleukin-1 (IL-1) secretion in vitro in dendritic cells and macrophages via Nucleotide-binding domain and leucinerich repeat-containing (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome activation. However, the contribution of IL-1 to aluminduced innate and adaptive immune responses is controversial and the role of IL-1a following alum injection has not been addressed. This study shows that IL-1 is dispensable for alum-induced antibody and CD8 T cell responses to ovalbumin. However, IL-1 is essential for neutrophil infiltration into the injection site, while recruitment of inflammatory monocytes and eosinophils is IL-1 independent. Both IL-1a and IL-1b are released at the site of injection and contribute to the neutrophil response. Surprisingly, these effects are NLRP3-inflammasome independent as is the infiltration of other cell populations. However, while NLRP3 and caspase 1 were dispensable, alum-induced IL-1b at the injection site was dependent on the cysteine protease cathepsin S. Overall, these data demonstrate a previously unreported role for cathepsin S in IL-1b secretion, show that inflammasome formation is dispensable for alum-induced innate immunity and reveal that IL1a and IL-1b are both necessary for alum-induced neutrophil influx in vivo.
Obesity is characterized by chronic inflammation associated with neutrophil and M1 macrophage infiltration into white adipose tissue. However, the mechanisms underlying this process remain largely unknown. Based on the ability of oil-based adjuvants to induce immune responses, we hypothesized that endogenous oils derived from necrotic adipocytes may function as an immunological "danger signal." Here we show that endogenous oils of human origin are potent adjuvants, enhancing antibody responses to a level comparable to Freund's incomplete adjuvant. The endogenous oils were capable of promoting interleukin (IL)-1a-dependent recruitment of neutrophils and M1-like macrophages, while simultaneously diminishing M2-like macrophages. We found that endogenous oils from subcutaneous and omental adipocytes, and from healthy and unhealthy obese individuals, promoted comparable inflammatory responses. Furthermore, we also confirmed that white adipocytes in visceral fat of metabolically unhealthy obese (MUO) individuals are significantly larger than those in metabolically healthy obese individuals. Since adipocyte size is positively correlated with adipocyte death, we propose that endogenous oils have a higher propensity to be released from hypertrophied visceral fat in MUO individuals and that this is the key factor in driving inflammation. In summary, this study shows that adipocytes contain a potent oil adjuvant which drives IL1a-dependent proinflammatory responses in vivo.Obesity is characterized by chronic, low-grade inflammation resulting in insulin resistance. Understanding the mechanisms underlying this inflammation would provide valuable insights into the disease and potentially offer new therapeutic targets. Early studies showing that sodium salicylate can reverse the symptoms of type 2 diabetes (1) highlighted roles for inhibitor of kB kinase b (IKKb) and Jun N-terminal kinase (JNK) in this process. JNK can directly phosphorylate Serine307 on insulin receptor substrate 1, thus impairing insulin signaling and mediating obesity-induced insulin resistance (2). IKKb can also phosphorylate insulin receptor substrate 1 (3), and myeloid-specific deletion of IKKb in obese mice protects against insulin resistance (4). In addition, IKKbmediated translocation of nuclear factor-kB into the nucleus
Background: IAP antagonists sensitize toward apoptosis induced by TNF and other TNFR family ligands. Results: IAP antagonism exerted effects on spontaneous as well as TNF-induced cytokine and chemokine production. Conclusion: IAPs regulate spontaneous as well as TNF-induced cytokine/chemokine production. Significance: IAP antagonists modulate cytokine production as well as apoptosis, which could influence their utility as adjuncts to chemotherapy.
SummaryHumans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer.
Dendritic cell (DC) activation is commonly used as a measure of the immunomodulatory potential of candidate exogenous and endogenous molecules. Residual lipopolysaccharide (LPS) contamination is a recurring theme and the potency of LPS is not always fully appreciated. To address this, polymyxin B (PmB) is often used to neutralise contaminating LPS. However, the limited capacity of this antibiotic to successfully block these effects is neglected. Therefore, this study aimed to determine the minimum LPS concentration required to induce murine bone marrow-derived dendritic cell (BMDC) maturation and cytokine secretion and to assess the ability of PmB to inhibit these processes. LPS concentrations as low as 10 pg/ml and 20 pg/ml induced secretion of interleukin (IL)-6 and tumor necrosis factor (TNF)-α respectively, while a concentration of 50 pg/ml promoted secretion of IL-12p40. A much higher threshold exists for IL-12p70 as an LPS concentration of 500 pg/ml was required to induce secretion of this cytokine. The efficacy of PmB varied substantially for different cytokines but this antibiotic was particularly limited in its ability to inhibit LPS-induced secretion of IL-6 and TNF-α. Furthermore, an LPS concentration of 50 pg/ml was sufficient to promote DC expression of costimulatory molecules and PmB was limited in its capacity to reverse this process when LPS concentrations of greater than 20 ng/ml were used. There is a common perception that LPS is heat resistant. However, heat treatment attenuated the ability of low concentrations of LPS to induce secretion of IL-6 and IL-12p40 by BMDCs, thus suggesting that heat-inactivation of protein preparations is also an ineffective control for discounting potential LPS contamination. Finally, LPS concentrations of less than 10 pg/ml were incapable of promoting secretion of IL-6 independently but could synergise with heat-labile enterotoxin (LT) to promote IL-6, indicating that reducing contaminating endotoxin concentrations to low pg/ml concentrations is essential to avoid misleading conclusions regarding candidate immunomodulators.
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