SIRPα maintains macrophage homeostasis by interacting with PTK2B kinase in Mycobacterium tuberculosis infection and through autophagy and necroptosis.

BACKGROUND: To determine whether SIRPα can be a diagnostic marker of pulmonary tuberculosis (PTB) and the molecular mechanism of SIRPα regulating macrophages to kill Mycobacterium tuberculosis (MTB). METHODS: Meta-analysis combined with subsequent qRT-PCR, western-blotting and flow cytometry assay were used to detect SIRPα expression in PTB patients. Cell-based assays were used to explore the regulation of macrophage function by SIRPα. SIRPα-/- and wide type macrophages transplanted C57BL/6J mice were used to determine the function of SIRPα on MTB infection in vivo. FINDINGS: SIRPα levels are closely correlated with the treatment outcomes among PTB patients. Cell-based assay demonstrated that MTB significantly induces the expression of SIRPα on macrophages. SIRPα deficiency enhances the killing ability of macrophages against MTB through processes that involve enhanced autophagy and reduced necroptosis of macrophages. Mechanistically, SIRPα forms a direct interaction with PTK2B through its intracellular C-terminal domain, thus inhibiting PTK2B activation in macrophages. Necroptosis inhibition due to SIRPα deficiency requires PTK2B activity. The transfer of SIRPα-deficient bone marrow-derived macrophages (BMDMs) into wild type mice resulted in a drop of bacterial load in the lungs but an enhancement of inflammatory lung damage, and the combination of ulinastatin and SIRPα-/-→WT treatment could decrease the inflammation and maintain the bactericidal capacity. INTERPRETATION: Our data define SIRPα a novel biomarker for tuberculosis infection and underlying mechanisms for maintaining macrophage homeostasis. FUNDING: This work was financially supported by the Chinese National Natural Science Foundation project (No.81401635). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Immune dysregulation in SHARPIN-deficient mice is dependent on CYLD-mediated cell death.

SHARPIN, together with RNF31/HOIP and RBCK1/HOIL1, form the linear ubiquitin chain assembly complex (LUBAC) E3 ligase that catalyzes M1-linked polyubiquitination. Mutations in RNF31/HOIP and RBCK/HOIL1 in humans and Sharpin in mice lead to autoinflammation and immunodeficiency, but the mechanism underlying the immune dysregulation remains unclear. We now show that the phenotype of the Sharpincpdm/cpdm mice is dependent on CYLD, a deubiquitinase previously shown to mediate removal of K63-linked polyubiquitin chains. Dermatitis, disrupted splenic architecture, and loss of Peyer's patches in the Sharpincpdm/cpdm mice were fully reversed in Sharpincpdm/cpdm Cyld-/- mice. We observed enhanced association of RIPK1 with the death-signaling Complex II following TNF stimulation in Sharpincpdm/cpdm cells, a finding dependent on CYLD since we observed reversal in Sharpincpdm/cpdm Cyld-/- cells. Enhanced RIPK1 recruitment to Complex II in Sharpincpdm/cpdm cells correlated with impaired phosphorylation of CYLD at serine 418, a modification reported to inhibit its enzymatic activity. The dermatitis in the Sharpincpdm/cpdm mice was also ameliorated by the conditional deletion of Cyld using LysM-cre or Cx3cr1-cre indicating that CYLD-dependent death of myeloid cells is inflammatory. Our studies reveal that under physiological conditions, TNF- and RIPK1-dependent cell death is suppressed by the linear ubiquitin-dependent inhibition of CYLD. The Sharpincpdm/cpdm phenotype illustrates the pathological consequences when CYLD inhibition fails.

Human TBK1 deficiency leads to autoinflammation driven by TNF-induced cell death.

TANK binding kinase 1 (TBK1) regulates IFN-I, NF-κB, and TNF-induced RIPK1-dependent cell death (RCD). In mice, biallelic loss of TBK1 is embryonically lethal. We discovered four humans, ages 32, 26, 7, and 8 from three unrelated consanguineous families with homozygous loss-of-function mutations in TBK1. All four patients suffer from chronic and systemic autoinflammation, but not severe viral infections. We demonstrate that TBK1 loss results in hypomorphic but sufficient IFN-I induction via RIG-I/MDA5, while the system retains near intact IL-6 induction through NF-κB. Autoinflammation is driven by TNF-induced RCD as patient-derived fibroblasts experienced higher rates of necroptosis in vitro, and CC3 was elevated in peripheral blood ex vivo. Treatment with anti-TNF dampened the baseline circulating inflammatory profile and ameliorated the clinical condition in vivo. These findings highlight the plasticity of the IFN-I response and underscore a cardinal role for TBK1 in the regulation of RCD.

Reversal of CYLD phosphorylation as a novel therapeutic approach for adult T-cell leukemia/lymphoma (ATLL).

Adult T-cell leukemia/lymphoma (ATLL) is a malignancy of mature T cells associated with chronic infection by human T-cell lymphotropic virus type-1 (HTLV-1). ATLL patients with aggressive subtypes have dismal outcomes. We demonstrate that ATLL cells co-opt an early checkpoint within the tumor necrosis factor receptor 1 (TNFR1) pathway, resulting in survival advantage. This early checkpoint revolves around an interaction between the deubiquitinase CYLD and its target RIPK1. The status of RIPK1 K63-ubiquitination determines cell fate by creating either a prosurvival signal (ubiquitinated RIPK1) or a death signal (deubiquitinated RIPK1). In primary ATLL samples and in cell line models, an increased baseline level of CYLD phosphorylation was observed. We therefore tested the hypothesis that this modification of CYLD, which has been reported to inhibit its deubiquitinating function, leads to increased RIPK1 ubiquitination and thus provides a prosurvival signal to ATLL cells. CYLD phosphorylation can be pharmacologically reversed by IKK inhibitors, specifically by TBK1/IKKε and IKKß inhibitors (MRT67307 and TPCA). Both of the IKK sub-families can phosphorylate CYLD, and the combination of MRT67307 and TPCA have a marked effect in reducing CYLD phosphorylation and triggering cell death. ATLL cells overexpressing a kinase-inactive TBK1 (TBK1-K38A) demonstrate lower CYLD phosphorylation and subsequently reduced proliferation. IKK blockade reactivates CYLD, as evidenced by the reduction in RIPK1 ubiquitination, which leads to the association of RIPK1 with the death-inducing signaling complex (DISC) to trigger cell death. In the absence of CYLD, RIPK1 ubiquitination remains elevated following IKK blockade and it does not associate with the DISC. SMAC mimetics can similarly disrupt CYLD phosphorylation and lead to ATLL cell death through reduction of RIPK1 ubiquitination, which is CYLD dependent. These results identify CYLD as a crucial regulator of ATLL survival and point to its role as a potential novel target for pharmacologic modification in this disease.

Single-Cell and Population-Level Analyses Using Real-Time Kinetic Labeling Couples Proliferation and Cell Death Mechanisms.

Quantifying cytostatic and cytotoxic outcomes are integral components of characterizing perturbagens used as research tools and in drug discovery pipelines. Furthermore, data-rich acquisition, coupled with robust methods for analysis, is required to properly assess the function and impact of these perturbagens. Here, we present a detailed and versatile method for single-cell and population-level analyses using real-time kinetic labeling (SPARKL). SPARKL integrates high-content live-cell imaging with automated detection and analysis of fluorescent reporters of cell death. We outline several examples of zero-handling, non-disruptive protocols for detailing cell death mechanisms and proliferation profiles. Additionally, we suggest several methods for mathematically analyzing these data to best utilize the collected kinetic data. Compared to traditional methods of detection and analysis, SPARKL is more sensitive, accurate, and high throughput while substantially eliminating sample processing and providing richer data.

Interleukin-1ß-induced IRAK1 ubiquitination is required for TH-GM-CSF cell differentiation in T cell-mediated inflammation.

Accumulating evidence suggests granulocyte macrophage-colony stimulating factor (GM-CSF) can function as an inflammatory mediator, but whether GM-CSF-producing CD4+ T cells (TH-GM-CSF) are a distinct T helper cell subset is lacking. Herein we demonstrate that interleukin (IL)-1ß exclusively drives differentiation of naïve CD4+ T cells into TH-GM-CSF cells via inducing ubiquitination of IL-1 receptor-associated kinase 1 (IRAK1) and subsequent activation of the transcription factor NF-kappaB (NF-κB), independent of RAR-related orphan receptor gamma (RORγt) required for TH17 differentiation. In vivo, TH-GM-CSF cells are present in murine Citrobacter Rodentium infections and mediate colitis following adoptive transfer of CD4+ T cells into Rag1-/- mice via GM-CSF-induced macrophage activation. The TH-GM-CSF cell phenotype is stable and distinct from the TH17 genetic program, but IL-1ß can convert pre-formed TH17 cells into TH-GM-CSF cells, thereby accounting for previously reported associations between IL-17 and GM-CSF. Together, our results newly identify IL-1ß/NF-κB-dependent TH-GM-CSF cells as a unique T helper cell subset and highlight the importance of CD4+ T cell-derived GM-CSF induced macrophage activation as a previously undescribed T cell effector mechanism.

Analysis of Necroptosis in Bone Marrow-Derived Macrophages.

Necroptosis in macrophages is increasingly being recognized as a significant contributor to the inflammation seen in many pathologies. Here we describe a well-known method to obtain quiescent, mature macrophages that can be used to study the molecular mechanisms that regulate necroptosis. We also describe two ways to quantify this form of programmed cell death.

Analysis of CYLD Proteolysis by CASPASE 8 in Bone Marrow-Derived Macrophages.

Previous studies have demonstrated that CASPASE 8 can generate a prosurvival signal by inhibiting necroptosis via the cleavage of the deubiquitinating enzyme CYLD. Cleavage of CYLD at D215 results in the generation of a 25 kD N-terminal fragment and degradation of the C-terminal fragment containing the catalytic domain. Since CYLD is required for TNF-induced necroptosis, its proteolysis is necessary and sufficient to suppress necroptosis and generate a survival signal. Here we describe how to visualize CYLD proteolysis by western blot analysis, as a measure of CASPASE 8 activity and inhibition of necroptosis.

CYLD Proteolysis Protects Macrophages from TNF-Mediated Auto-necroptosis Induced by LPS and Licensed by Type I IFN.

Tumor necrosis factor (TNF) induces necroptosis, a RIPK3/MLKL-dependent form of inflammatory cell death. In response to infection by Gram-negative bacteria, multiple receptors on macrophages, including TLR4, TNF, and type I IFN receptors, are concurrently activated, but it is unclear how they crosstalk to regulate necroptosis. We report that TLR4 activates CASPASE-8 to cleave and remove the deubiquitinase cylindromatosis (CYLD) in a TRIF- and RIPK1-dependent manner to disable necroptosis in macrophages. Inhibiting CASPASE-8 leads to CYLD-dependent necroptosis caused by the TNF produced in response to TLR4 ligation. While lipopolysaccharides (LPS)-induced necroptosis was abrogated in Tnf(-/-) macrophages, a soluble TNF antagonist was not able to do so in Tnf(+/+) macrophages, indicating that necroptosis occurs in a cell-autonomous manner. Surprisingly, TNF-mediated auto-necroptosis of macrophages requires type I IFN, which primes the expression of key necroptosis-signaling molecules, including TNFR2 and MLKL. Thus, the TNF necroptosis pathway is regulated by both negative and positive crosstalk.

NEMO inhibits programmed necrosis in an NFκB-independent manner by restraining RIP1.

TNF can trigger two opposing responses: cell survival and cell death. TNFR1 activates caspases that orchestrate apoptosis but some cell types switch to a necrotic death when treated with caspase inhibitors. Several genes that are required to orchestrate cell death by programmed necrosis have been identified, such as the kinase RIP1, but very little is known about the inhibitory signals that keep this necrotic cell death pathway in check. We demonstrate that T cells lacking the regulatory subunit of IKK, NFκB essential modifier (NEMO), are hypersensitive to programmed necrosis when stimulated with TNF in the presence of caspase inhibitors. Surprisingly, this pro-survival activity of NEMO is independent of NFκB-mediated gene transcription. Instead, NEMO inhibits necrosis by binding to ubiquitinated RIP1 to restrain RIP1 from engaging the necrotic death pathway. In the absence of NEMO, or if ubiquitination of RIP1 is blocked, necrosis ensues when caspases are blocked. These results indicate that recruitment of NEMO to ubiquitinated RIP1 is a key step in the TNFR1 signaling pathway that determines whether RIP1 triggers a necrotic death response.

Suppression of NF-kappaB-mediated beta-defensin gene expression in the mammalian airway by the Bordetella type III secretion system.

Expression of innate immune genes such as beta-defensins is induced in airway epithelium by bacterial components via activation of NF-kappaB. We show here that live Gram-negative bacteria can similarly stimulate this pathway, resulting in upregulation of the beta-defensin tracheal antimicrobial peptide (TAP) in primary cultures of bovine tracheal epithelial cells (TECs), by a Toll-like receptor 4 (TLR4)-mediated pathway. The Gram-negative airway pathogen Bordetella bronchiseptica possesses a type III secretion system previously suggested to inhibit the nuclear translocation of NF-kappaB in a cell line by immunohistochemistry. We therefore hypothesized that this pathogen might interfere in the innate immune response of the epithelium. Exposure of TECs to wild-type B. bronchiseptica suppressed the activation of NF-kappaB and the subsequent induction of TAP mRNA levels, whereas a type III secretion-defective strain did not. These results suggest a mechanism for bacterial evasion of the innate immune response in the airway, which could allow for the observed persistent colonization of this pathogen.

One of two human lactoferrin variants exhibits increased antibacterial and transcriptional activation activities and is associated with localized juvenile periodontitis.

The iron-binding protein lactoferrin is a ubiquitous and abundant constituent of human exocrine secretions. Lactoferrin inhibits bacterial growth by sequestering essential iron and also exhibits non-iron-dependent antibacterial, antifungal, antiviral, antitumor, anti-inflammatory, and immunoregulatory activities. All of these non-iron-dependent activities are mediated by the highly charged N terminus of lactoferrin. In this study we characterized a Lys/Arg polymorphism at position 29 in the N-terminal region of human lactoferrin that results from a single nucleotide polymorphism in exon 1 of the human lactoferrin gene. We expressed cDNAs encoding both lactoferrin variants in insect cells and purified the two proteins by ion exchange chromatography. The two lactoferrin variants exhibited nearly identical iron-binding and iron-releasing activities and equivalent bactericidal activities against a strain of the gram-negative bacterium Actinobacillus actinomycetemcomitans. When tested against the gram-positive species Streptococcus mutans and Streptococcus mitis, however, lactoferrin containing Lys at position 29 exhibited significantly greater bactericidal activity than did lactoferrin containing Arg. In addition, the Lys-containing lactoferrin stimulated bovine tracheal epithelial cells to synthesize much higher levels of tracheal antimicrobial peptide mRNA than did the Arg-containing variant. A genotyping assay that distinguished between the two alleles based on a polymorphic EarI restriction site showed that the Lys and Arg alleles had frequencies of 24% and 76%, respectively, among 17 healthy human subjects, and 72% and 28%, respectively, among nine patients with localized juvenile periodontitis. Our findings suggest that these two lactoferrin variants are functionally different and that these differences may contribute to the pathogenesis of localized juvenile periodontitis.