Asymmetric Engagement of Dimeric CRL3 KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes.

UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal, a process that is tightly controlled by epigenetic regulation. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1 to promote the degradation of select HDAC1/2 corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KBTBD4 KELCH-repeat propeller domains to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.

DrugMap: A quantitative pan-cancer analysis of cysteine ligandability.

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed "DrugMap," an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity.

DrugMap: A quantitative pan-cancer analysis of cysteine ligandability.

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors of a wide-range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed DrugMap , an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NFκB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NFκB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription factor activity.

Systematic identification of anticancer drug targets reveals a nucleus-to-mitochondria ROS-sensing pathway.

Multiple anticancer drugs have been proposed to cause cell death, in part, by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs, exactly how the resultant ROS function and are sensed is poorly understood. It remains unclear which proteins the ROS modify and their roles in drug sensitivity/resistance. To answer these questions, we examined 11 anticancer drugs with an integrated proteogenomic approach identifying not only many unique targets but also shared ones-including ribosomal components, suggesting common mechanisms by which drugs regulate translation. We focus on CHK1 that we find is a nuclear H2O2 sensor that launches a cellular program to dampen ROS. CHK1 phosphorylates the mitochondrial DNA-binding protein SSBP1 to prevent its mitochondrial localization, which in turn decreases nuclear H2O2. Our results reveal a druggable nucleus-to-mitochondria ROS-sensing pathway-required to resolve nuclear H2O2 accumulation and mediate resistance to platinum-based agents in ovarian cancers.

Identification of chemotherapy targets reveals a nucleus-to-mitochondria ROS sensing pathway.

Multiple chemotherapies are proposed to cause cell death in part by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs exactly how the resultant ROS function and are sensed is poorly understood. In particular, it's unclear which proteins the ROS modify and their roles in chemotherapy sensitivity/resistance. To answer these questions, we examined 11 chemotherapies with an integrated proteogenomic approach identifying many unique targets for these drugs but also shared ones including ribosomal components, suggesting one mechanism by which chemotherapies regulate translation. We focus on CHK1 which we find is a nuclear H 2 O 2 sensor that promotes an anti-ROS cellular program. CHK1 acts by phosphorylating the mitochondrial-DNA binding protein SSBP1, preventing its mitochondrial localization, which in turn decreases nuclear H 2 O 2 . Our results reveal a druggable nucleus-to-mitochondria ROS sensing pathway required to resolve nuclear H 2 O 2 accumulation, which mediates resistance to platinum-based chemotherapies in ovarian cancers.

NRF2 activation induces NADH-reductive stress, providing a metabolic vulnerability in lung cancer.

Multiple cancers regulate oxidative stress by activating the transcription factor NRF2 through mutation of its negative regulator, KEAP1. NRF2 has been studied extensively in KEAP1-mutant cancers; however, the role of this pathway in cancers with wild-type KEAP1 remains poorly understood. To answer this question, we induced NRF2 via pharmacological inactivation of KEAP1 in a panel of 50+ non-small cell lung cancer cell lines. Unexpectedly, marked decreases in viability were observed in >13% of the cell lines-an effect that was rescued by NRF2 ablation. Genome-wide and targeted CRISPR screens revealed that NRF2 induces NADH-reductive stress, through the upregulation of the NAD+-consuming enzyme ALDH3A1. Leveraging these findings, we show that cells treated with KEAP1 inhibitors or those with endogenous KEAP1 mutations are selectively vulnerable to Complex I inhibition, which impairs NADH oxidation capacity and potentiates reductive stress. Thus, we identify reductive stress as a metabolic vulnerability in NRF2-activated lung cancers.

Frequency-dependent hearing outcomes with or without preservation of intact ossicular articulations.

Objective: To determine the frequency-specific benefits of ossicular chain preservation compared to performing disarticulations and reconstructions in transmastoid facial nerve decompression surgery in patients with an intact ossicular chain. Methods: A retrospective chart review (January 2007 and June 2018) of patients undergoing transmastoid facial nerve decompression on the intact middle ear for severe facial palsy at a tertiary referral center. Surgery was performed with ossicular chain disarticulation on an as-needed basis using either ossicular chain preservation (without ossicular disarticulation), incudostapedial separation, or incus disarticulation technique. Hearing outcomes were assessed. Results: The 108 patients were included in this study. Among these, 89 patients underwent ossicular chain preservation, 5 underwent incudostapedial separation and 14 underwent incus repositioning. The proportion of patients with a change in the 4-frequency air conduction pure-tone average of less than 10 dB was 91%, 60%, and 50%, respectively, for the three surgical techniques; these were significantly different (Fisher's exact test, p < .001). Frequency-specific analysis showed that air conduction was significantly better following the ossicular chain preservation technique compared with the incus repositioning technique at stimulation frequencies lower than 250 Hz and higher than 2000 Hz, and compared with the incudostapedial separation technique at 4000 Hz. Analysis of biometric measures determined on CT images suggested that the feasibility of the ossicular chain preservation technique correlates with incus body thickness on coronal CT images. Conclusions: Ossicular chain preservation is an effective approach for hearing preservation in transmastoid facial nerve decompression or similar surgical procedures.

Acoustic neuromas associated with sudden sensorineural hearing loss.

BACKGROUND: Several studies have reported sudden sensorineural hearing loss (SHL) as an initial symptom of acoustic neuromas (ANs). AIMS/OBJECTIVES: We aimed to retrospectively evaluate the incidence of SHL and the associated rates of post-treatment improvement in patients diagnosed with ANs. MATERIALS AND METHODS: We analysed the clinical data of 686 patients with ANs and examined tumour size, correlation with hearing loss, number of episodes of SHL, extent of auditory recovery, and associated audiogram patterns. RESULTS: Among 686 enrolled patients with ANs, 232 (86 with intracanalicular and 146 with extrameatal tumours) experienced SHL at some point in their clinical history. The incidence of SHL was not significantly associated with tumour size. Of the 172 patients analysed, 119, 44, and 9 patients experienced one, two, or three or more episodes of SHL, respectively. Confirmed auditory recovery occurred in 61%, 45.3%, and 33.3% of the patients after the first, second, or third or later episodes of SHL, respectively. The audiograms of 78/172 (45.3%) patients showed a typical trough-shaped pattern of hearing loss at medium-level frequencies. CONCLUSIONS AND SIGNIFICANCE: Among patients with ANs, 7.7% experienced two or more episodes of SHL. The recovery rate of SHL decreased with each successive occurrence.

The tumor suppressor kinase DAPK3 drives tumor-intrinsic immunity through the STING-IFN-ß pathway.

Evasion of host immunity is a hallmark of cancer; however, mechanisms linking oncogenic mutations and immune escape are incompletely understood. Through loss-of-function screening of 1,001 tumor suppressor genes, we identified death-associated protein kinase 3 (DAPK3) as a previously unrecognized driver of anti-tumor immunity through the stimulator of interferon genes (STING) pathway of cytosolic DNA sensing. Loss of DAPK3 expression or kinase activity impaired STING activation and interferon (IFN)-ß-stimulated gene induction. DAPK3 deficiency in IFN-ß-producing tumors drove rapid growth and reduced infiltration of CD103+CD8α+ dendritic cells and cytotoxic lymphocytes, attenuating the response to cancer chemo-immunotherapy. Mechanistically, DAPK3 coordinated post-translational modification of STING. In unstimulated cells, DAPK3 inhibited STING K48-linked poly-ubiquitination and proteasome-mediated degradation. After cGAMP stimulation, DAPK3 was required for STING K63-linked poly-ubiquitination and STING-TANK-binding kinase 1 interaction. Comprehensive phospho-proteomics uncovered a DAPK3-specific phospho-site on the E3 ligase LMO7, critical for LMO7-STING interaction and STING K63-linked poly-ubiquitination. Thus, DAPK3 is an essential kinase for STING activation that drives tumor-intrinsic innate immunity and tumor immune surveillance.

Facial and hearing outcomes in transmastoid nerve decompression for Bell's palsy, with preservation of the ossicular chain.

OBJECTIVES: Facial nerve decompression is a salvage treatment for Bell's palsy patients for whom a poor prognosis is anticipated with standard medical treatment. The transmastoid approach is a frequently performed approach, but it remains unknown if this surgery is effective when the ossicular chain is preserved. This study aimed to determine the efficacy of facial nerve decompression using the transmastoid approach in Bell's palsy. DESIGN, SETTING AND PARTICIPANTS: This retrospective study included patients who had undergone transmastoid facial nerve decompression with ossicular chain preservation and patients who met the criteria for surgery, but received only medical treatment between January 2007 and May 2019, at a single centre. MAIN OUTCOME MEASURES: Attainment of House-Brackmann grade I at 12 months after onset of facial palsy. RESULTS: The recovery rate to House-Brackmann grade I in the decompression group in the early phase (≤18 days after onset) was higher than that of the medical treatment group, although the difference was not significant (70% vs 47%, P = .160). However, within this early surgery group, a subgroup of cases with ≥95% facial nerve degeneration demonstrated a significant improvement in recovery rate (73% vs 30%, P = .018). Among surgeries performed in the late phase (≥19 days), only a subgroup with ≥95% facial nerve degeneration was available for analysis, and the difference in recovery rate was not significant compared with medical treatment alone (26% vs 30%, P = 1.00). Post-surgical hearing evaluation demonstrated that average hearing deterioration was 1.3 dB which was non-significant, suggesting this procedure does not cause hearing loss. CONCLUSIONS: Transmastoid facial nerve decompression with ossicular chain preservation in the early phase after symptom-onset is an effective salvage treatment for severe Bell's palsy with ≥95% facial nerve degeneration.

Laparoscopic-Assisted Hysteroscopic Surgery for Endometrial Tumors Arising after Pelvic Radiation Therapy.

Uterine tumors detected after pelvic radiation therapy are rare. We report a case in which an endometrial tumor developed after pelvic radiation therapy for cervical cancer. The patient was a 70-year-old female with a history of pelvic radiotherapy for locally advanced cervical cancer. After 12 months of radiation therapy, magnetic resonance imaging showed tumors in the uterine cavity, and positron emission tomography-computed tomography showed no tumors, except uterine tumors. Since radiation therapy-induced cervical stenosis was conducted, endometrial examination could not be performed without anesthesia. As these tumors were detected after radiation therapy for uterine cancer, malignancy was considered. A laparoscopic-assisted transcervical hysteroscopic resection was performed for the diagnosis and treatment of uterine tumors after radiation therapy. This operative method was useful and enabled us to perform complete resection, observe the abdominal cavity, prepare for the possibility of secondary hysterectomy, and improve safety.

Cellular sensing of extracellular purine nucleosides triggers an innate IFN-ß response.

Mechanisms linking immune sensing of DNA danger signals in the extracellular environment to innate pathways in the cytosol are poorly understood. Here, we identify a previously unidentified immune-metabolic axis by which cells respond to purine nucleosides and trigger a type I interferon-ß (IFN-ß) response. We find that depletion of ADA2, an ectoenzyme that catabolizes extracellular dAdo to dIno, or supplementation of dAdo or dIno stimulates IFN-ß. Under conditions of reduced ADA2 enzyme activity, dAdo is transported into cells and undergoes catabolysis by the cytosolic isoenzyme ADA1, driving intracellular accumulation of dIno. dIno is a functional immunometabolite that interferes with the cellular methionine cycle by inhibiting SAM synthetase activity. Inhibition of SAM-dependent transmethylation drives epigenomic hypomethylation and overexpression of immune-stimulatory endogenous retroviral elements that engage cytosolic dsRNA sensors and induce IFN-ß. We uncovered a previously unknown cellular signaling pathway that responds to extracellular DNA-derived metabolites, coupling nucleoside catabolism by adenosine deaminases to cellular IFN-ß production.

Clinical and Radiological Characteristics of Malignant Tumors Located to the Cerebellopontine Angle and/or Internal Acoustic Meatus.

OBJECTIVE: Metastatic lesions to the internal auditory meatus (IAM) and/or the cerebellopontine angle (CPA) are rare and may appear like a vestibular schwannoma (VS). We herein raise the issue of the diagnosis and treatment of nine malignant cases of the CPA and IAM among three referral centers in France and Japan. The aim of this study was 1) to report malignant lesions of the CPA, their diagnosis and treatment, 2) to review the literature, 3) to propose criteria of suspicion for malignant tumors of the CPA. METHODS: Nine patients who had malignant lesions of the CPA and/or IAM for whom the final diagnosis was made by surgery, lumbar puncture, or PET scan were included. The main outcomes measured were: rapid onset of symptoms, association of cochlea-vestibular symptoms with facial palsy, and MRI analysis. RESULTS: Among the nine patients with malignant tumor of the CPA, 8 of them (89%) had a facial palsy associated with cochlea-vestibular symptoms. Rapid growth of the tumor was observed in 77% (7/9) of the cases in a mean time interval of 4.6 months. The initial diagnosis evoked was VS in 44% of the cases (4/9). Atypical MRI aspect was seen in 67% of the cases (6/9) with bilateral tumors in 55% of cases (5/9). CONCLUSION: Although rare, malignant tumors of the CPA and/or IAM should be evoked in case of association of cochleovestibular symptoms and facial palsy, rapid onset and atypical MRI aspect.

Developmental delays assessed using the Enjoji Scale in children with cochlear implants who have intellectual disability with or without autism spectrum disorder.

OBJECTIVE: Intellectual disability (ID) and autism spectrum disorder (ASD) are common among children who are candidates for cochlear implants. However, the implications of these comorbidities for cochlear implant placement have been not fully established. This study sought to identify these implications by comparing developmental delays among children with these conditions. METHODS: Participants were children who were followed up at least every 6 months for 24 months after cochlear implant surgery. Developmental delays were assessed using the Enjoji Scale of Infant Analytical Development (Enjoji Scale) and compared in three groups with hearing loss: those with ID (ID group, n=4); those with ASD and ID (ASD+ID group, n=4); and those with typical development (control group, n=5). Developmental delay was evaluated longitudinally before and after cochlear implant placement for 18 months. RESULTS: Among the six subscales that make up the Enjoji Scale, language development and intelligence development were significantly delayed in all three groups and were exacerbated over time except for language development in the control group. Emotional development and social behavior were significantly delayed only in the ASD+ID group. Comparison of intergroup differences revealed delays in language development in the ID and ASD+ID groups compared with the control group. CONCLUSION: The Enjoji Scale successfully demonstrated developmental delays characteristic to the underlying comorbidities of ID with or without ASD in children with cochlear implants. The Enjoji Scale can be a useful diagnostic tool for screening children with cochlear implants for ID with or without ASD.

Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8)/CLM-6 is an FcRγ-coupled receptor selectively expressed in mouse tissue plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) produce large amounts of type-I interferon (IFN) in response to viral infection or self nucleic acids. Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8), also called CMRF-35-like molecule-6 (CLM-6), is a putative activating receptor among mouse LMIR/CLM/CD300 members; however, the expression and function of LMIR8 remain unclear. Here, we characterize mouse LMIR8 as a pDC receptor. Analysis of Flag-tagged LMIR8-transduced bone marrow (BM)-derived mast cells demonstrated that LMIR8 can transmit an activating signal by interacting with immunoreceptor tyrosine-based activating motif (ITAM)-containing FcRγ. Flow cytometric analysis using a specific antibody for LMIR8 showed that LMIR8 expression was restricted to mouse pDCs residing in BM, spleen, or lymph node. FcRγ deficiency dampened surface expression of LMIR8 in mouse pDCs. Notably, LMIR8 was detected only in pDCs, irrespective of TLR9 stimulation, suggesting that LMIR8 is a suitable marker for pDCs in mouse tissues; LMIR8 is weakly expressed in Flt3 ligand-induced BM-derived pDCs (BMpDCs). Crosslinking of transduced LMIR8 in BMpDCs with anti-LMIR8 antibody did not induce IFN-α production, but rather suppressed TLR9-mediated production of IFN-α. Taken together, these observations indicate that LMIR8 is an FcRγ-coupled receptor selectively expressed in mouse tissue pDCs, which might suppress pDC activation through the recognition of its ligands.

The CD300e molecule in mice is an immune-activating receptor.

CD300 molecules (CD300s) belong to paired activating and inhibitory receptor families, which mediate immune responses. Human CD300e (hCD300e) is expressed in monocytes and myeloid dendritic cells and transmits an immune-activating signal by interacting with DNAX-activating protein 12 (DAP12). However, the CD300e ortholog in mice (mCD300e) is poorly characterized. Here, we found that mCD300e is also an immune-activating receptor. We found that mCD300e engagement triggers cytokine production in mCD300e-transduced bone marrow-derived mast cells (BMMCs). Loss of DAP12 and another signaling protein, FcRγ, did not affect surface expression of transduced mCD300e, but abrogated mCD300e-mediated cytokine production in the BMMCs. Co-immunoprecipitation experiments revealed that mCD300e physically interacts with both FcRγ and DAP12, suggesting that mCD300e delivers an activating signal via these two proteins. Binding and reporter assays with the mCD300e extracellular domain identified sphingomyelin as a ligand of both mCD300e and hCD300e. Notably, the binding of sphingomyelin to mCD300e stimulated cytokine production in the transduced BMMCs in an FcRγ- and DAP12-dependent manner. Flow cytometric analysis with an mCD300e-specific Ab disclosed that mCD300e expression is highly restricted to CD115+Ly-6Clow/int peripheral blood monocytes, corresponding to CD14dim/+CD16+ human nonclassical and intermediate monocytes. Loss of FcRγ or DAP12 lowered the surface expression of endogenous mCD300e in the CD115+Ly-6Clow/int monocytes. Stimulation with sphingomyelin failed to activate the CD115+Ly-6Clow/int mouse monocytes, but induced hCD300e-mediated cytokine production in the CD14dimCD16+ human monocytes. Taken together, these observations indicate that mCD300e recognizes sphingomyelin and thereby regulates nonclassical and intermediate monocyte functions through FcRγ and DAP12.

Cytosolic sensing of immuno-stimulatory DNA, the enemy within.

In the cytoplasm, DNA is sensed as a universal danger signal by the innate immune system. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor/enzyme that catalyzes formation of 2'-5'-cGAMP, an atypical cyclic di-nucleotide second messenger that binds and activates the Stimulator of Interferon Genes (STING), resulting in recruitment of Tank Binding Kinase 1 (TBK1), activation of the transcription factor Interferon Regulatory Factor 3 (IRF3), and trans-activation of innate immune response genes, including type I Interferon cytokines (IFN-I). Activation of the pro-inflammatory cGAS-STING-IRF3 response is triggered by direct recognition of the DNA genomes of bacteria and viruses, but also during RNA virus infection, neoplastic transformation, tumor immunotherapy and systemic auto-inflammatory diseases. In these circumstances, the source of immuno-stimulatory DNA has often represented a fundamental yet poorly understood aspect of the response. This review focuses on recent findings related to cGAS activation by an array of self-derived DNA substrates, including endogenous retroviral elements, mitochondrial DNA (mtDNA) and micronuclei generated as a result of genotoxic stress and DNA damage. These findings emphasize the role of the cGAS axis as a cell-intrinsic innate immune response to a wide variety of genomic insults.

Postoperative Headache after Undergoing Acoustic Neuroma Surgery via the Retrosigmoid Approach.

To estimate the duration of postoperative headache after surgery for acoustic neuroma and the effects of age, sex, tumor size, extent of tumor resection, type of skin incision, surgical duration, hearing preservation, and postoperative facial nerve palsy. This retrospective review analyzed clinical data from 97 patients who had undergone surgery for unilateral acoustic neuroma via the retrosigmoid approach >1 year previously. We investigated whether patients had headache at hospital discharge and during attendance at outpatient clinics. We classified postoperative headache as grade 0 (no headache), 1 (tolerable headache without medication), or 2 (headache requiring medication). The period of headache was defined as the interval in days between surgery and achievement of grade 0. The period of medication for headache was defined as the interval in days between surgery and achievement of grade 0 or 1. Kaplan-Meier analysis revealed median durations of medication and headache of 81 and 641 days, respectively. Headache was cured significantly earlier in patients who underwent surgery using a C-type skin incision (P < 0.001). Headache persisted significantly longer among patients who underwent a shorter surgical procedure (P < 0.02). Multivariate analysis confirmed the type of skin incision as a factor independently associated with duration of postoperative headache. Postoperative headache was cured in the majority of patients within about 2 years after surgery. The C-type skin incision is likely beneficial for reducing the duration of postoperative headache, although headache persisted in a small number of patients.

Disrupting ceramide-CD300f interaction prevents septic peritonitis by stimulating neutrophil recruitment.

Sepsis is a serious clinical problem. Negative regulation of innate immunity is associated with sepsis progression, but the underlying mechanisms remains unclear. Here we show that the receptor CD300f promotes disease progression in sepsis. CD300f -/- mice were protected from death after cecal ligation and puncture (CLP), a murine model of septic peritonitis. CD300f was highly expressed in mast cells and recruited neutrophils in the peritoneal cavity. Analysis of mice (e.g., mast cell-deficient mice) receiving transplants of wild-type or CD300f -/- mast cells or neutrophils indicated that CD300f deficiency did not influence intrinsic migratory abilities of neutrophils, but enhanced neutrophil chemoattractant production (from mast cells and neutrophils) in the peritoneal cavity of CLP-operated mice, leading to robust accumulation of neutrophils which efficiently eliminated Escherichia coli. Ceramide-CD300f interaction suppressed the release of neutrophil chemoattractants from Escherichia coli-stimulated mast cells and neutrophils. Administration of the reagents that disrupted the ceramide-CD300f interaction prevented CLP-induced sepsis by stimulating neutrophil recruitment, whereas that of ceramide-containing vesicles aggravated sepsis. Extracellular concentrations of ceramides increased in the peritoneal cavity after CLP, suggesting a possible role of extracellular ceramides, CD300f ligands, in the negative-feedback suppression of innate immune responses. Thus, CD300f is an attractive target for the treatment of sepsis.