CEACAM1 Negatively Regulates IL-1β Production in LPS Activated Neutrophils by Recruiting SHP-1 to a SYK-TLR4-CEACAM1 Complex

LPS-activated neutrophils secrete IL-1β by activation of TLR-4. Based on studies in macrophages, it is likely that ROS and lysosomal destabilization regulated by Syk activation may also be involved. Since neutrophils have abundant expression of the ITIM-containing co-receptor CEACAM1 and Gram-negative bacteria such as Neisseria utilize CEACAM1 as a receptor that inhibits inflammation, we hypothesized that the overall production of IL-1β in LPS treated neutrophils may be negatively regulated by CEACAM1. We found that LPS treated neutrophils induced phosphorylation of Syk resulting in the formation of a complex including TLR4, p-Syk, and p-CEACAM1, which in turn, recruited the inhibitory phosphatase SHP-1. LPS treatment leads to ROS production, lysosomal damage, caspase-1 activation and IL-1β secretion in neutrophils. The absence of this regulation in Ceacam1−/− neutrophils led to hyper production of IL-1β in response to LPS. The hyper production of IL-1β was abrogated by in vivo reconstitution of wild type but not ITIM-mutated CEACAM1 bone marrow stem cells. Blocking Syk activation by kinase inhibitors or RNAi reduced Syk phosphorylation, lysosomal destabilization, ROS production, and caspase-1 activation in Ceacam1−/− neutrophils. We conclude that LPS treatment of neutrophils triggers formation of a complex of TLR4 with pSyk and pCEACAM1, which upon recruitment of SHP-1 to the ITIMs of pCEACAM1, inhibits IL-1β production by the inflammasome. Thus, CEACAM1 fine-tunes IL-1β production in LPS treated neutrophils, explaining why the additional utilization of CEACAM1 as a pathogen receptor would further inhibit inflammation

Correction to: Toward a comprehensive view of cancer immune responsiveness: a synopsis from the SITC workshop.

Following publication of the original article [1], the author reported that an author name, Roberta Zappasodi, was missed in the authorship list

Toward a comprehensive view of cancer immune responsiveness: a synopsis from the SITC workshop.

Tumor immunology has changed the landscape of cancer treatment. Yet, not all patients benefit as cancer immune responsiveness (CIR) remains a limitation in a considerable proportion of cases. The multifactorial determinants of CIR include the genetic makeup of the patient, the genomic instability central to cancer development, the evolutionary emergence of cancer phenotypes under the influence of immune editing, and external modifiers such as demographics, environment, treatment potency, co-morbidities and cancer-independent alterations including immune homeostasis and polymorphisms in the major and minor histocompatibility molecules, cytokines, and chemokines. Based on the premise that cancer is fundamentally a disorder of the genes arising within a cell biologic process, whose deviations from normality determine the rules of engagement with the host\u27s response, the Society for Immunotherapy of Cancer (SITC) convened a task force of experts from various disciplines including, immunology, oncology, biophysics, structural biology, molecular and cellular biology, genetics, and bioinformatics to address the complexity of CIR from a holistic view. The task force was launched by a workshop held in San Francisco on May 14-15, 2018 aimed at two preeminent goals: 1) to identify the fundamental questions related to CIR and 2) to create an interactive community of experts that could guide scientific and research priorities by forming a logical progression supported by multiple perspectives to uncover mechanisms of CIR. This workshop was a first step toward a second meeting where the focus would be to address the actionability of some of the questions identified by working groups. In this event, five working groups aimed at defining a path to test hypotheses according to their relevance to human cancer and identifying experimental models closest to human biology, which include: 1) Germline-Genetic, 2) Somatic-Genetic and 3) Genomic-Transcriptional contributions to CIR, 4) Determinant(s) of Immunogenic Cell Death that modulate CIR, and 5) Experimental Models that best represent CIR and its conversion to an immune responsive state. This manuscript summarizes the contributions from each group and should be considered as a first milestone in the path toward a more contemporary understanding of CIR. We appreciate that this effort is far from comprehensive and that other relevant aspects related to CIR such as the microbiome, the individual\u27s recombined T cell and B cell receptors, and the metabolic status of cancer and immune cells were not fully included. These and other important factors will be included in future activities of the taskforce. The taskforce will focus on prioritization and specific actionable approach to answer the identified questions and implementing the collaborations in the follow-up workshop, which will be held in Houston on September 4-5, 2019

Model for the inhibition of the inflammasome in neutrophils by CEACAM1.

<p>(<b>A</b>) LPS binds to TLR (the usual downstream effects such as activation of NFκB are not shown for clarity). (<b>B</b>) The complex recruits and activates Syk (pSyk) which in the absence of CEACAM1 fully activates the inflammasome that includes ROS production from the mitochondrion and cathepsin B from the lysosome. The activated inflammasome converts pro-caspase-1 to active caspase-1, which in turn converts pro-IL-1β to active IL-1β. (<b>C</b>) In the presence of CEACAM1, both Syk and CEACAM1 are phosphorylated when LPS binds to TLR4. CEACAM1 recruits SHP1 via its phosphorylated ITIM. SHP1 dephosphorylates pSyk, reducing the production of ROS and lysosome disruption, which in turn, reduces the activity of the inflammasome.</p

Loss of CEACAM1 leads to elevated Syk activation and enhanced inflammasome activation.

<p>(<b>A</b>) Immunoblot analysis of WT and Ceacam1^−/− (KO) neutrophils with p-Syk (Y525/526), Syk and GAPDH antibodies. (<b>B</b>) Immunoblot analysis of TLR4, Syk, p-Syk and CEACAM1 in WT and Ceacam1^−/− (KO) neutrophils with or without LPS treatment (100 ng/ml) after immunoprecipitation with TLR4 antibody. (<b>C</b>) Confocal microscopy showing LPS induced lysosomal destabilization in WT and Ceacam1^−/− neutrophils (DQ-Ovalbumin, 10 mg/ml; green) with or without LPS (100 ng/ml), cell membranes were stained with fluorescent cholera toxin B-subunit (red). (<b>D</b>) Quantification of lysosomal destabilization of WT and Ceacam1^−/− neutrophils as measured by MFI of LysoSensor Green using FACS. (<b>E</b>) IL-1β production in the supernatants of LPS treated WT and Ceacam1^−/− neutrophils under different treatment conditions. (<b>F</b>) Immunoblot analysis showing caspase-1 activation of LPS treated WT and Ceacam1^−/− neutrophils under different treatment conditions. (<b>G</b>) IL-1β production in the supernatants of LPS treated WT neutrophils with or without glibenclamide treatment (250 µM). Data are representative of 3 different experiments and p values (<b>C</b>) were calculated by a 2-tailed T-test *0.01</p

LPS triggered IL-1β production in WT and Ceacam1^−/− neutrophils is partially dependent on cathepsin B.

<p>(<b>A</b>) Confocal microscopy showing cathepsin B activity in LPS treated WT and Ceacam1^−/− (KO) neutrophils. (<b>B</b>) Immunoblot analysis showing cathepsin B amounts in the supernatants of LPS treated WT and Ceacam1^−/− neutrophils. (<b>C</b>) IL-1β production in the supernatants of LPS treated WT and Ceacam1^−/− neutrophils after treatment with CA-074-Me. (<b>D</b>) Immunoblot analysis showing caspase-1 activation of LPS treated WT and Ceacam1^−/− neutrophils after CA-074-Me treatment. Data are representative of 3 different experiments (<b>A</b>, <b>B</b> and <b>D</b>) and p values (<b>C</b>) were calculated by a 2-tailed T-test ***p≤0.001.</p

LPS induced neutrophil inflammasome activation depends on ROS production and lysosome destabilization.

<p>(<b>A</b>) IL-1β production in the supernatants of wild type (WT), P2X7^−/− and Cybb^−/− neutrophils under different treatment conditions: LPS (100 ng/ml), KN62 (2 µM, neutrophils were pre-treated for 30 minutes before LPS treatment), APDC (100 µM), bafilomycin A (125 nM), z-YVAD-fmk (1 mM). (<b>B</b>) Immunoblot analysis showing caspase-1 activation of WT, P2X7^−/− and Cybb^−/− neutrophils under different treatment conditions. (<b>C</b>) ROS production by LPS treated WT and CEACAM1^−/− neutrophils with or without APDC (100 µM) as measured by MFI of fluorescent probe H2DCFDA using FACS. Data are representative of 3 different experiments (<b>B</b>) and p values (<b>A</b> and <b>C</b>) were calculated by a 2-tailed T-test, **0.001</p

CEACAM1 down-regulates Syk activation through ITIM recruitment of SHP-1.

<p>(<b>A</b>) Immunoblot analysis of p-Tyr, Syk, SHP-1 and CEACAM1 in WT and Ceacam1^−/− (KO) neutrophils with or without LPS treatment (100 ng/ml) after IP with anti-CEACAM1 antibody. (<b>B</b>) Immunoblot analysis of Syk, SHP-1 in WT and Ceacam1^−/− neutrophils with or without LPS after IP with anti-SHP-1 antibody. (<b>C</b>) Immunoblot analysis of p-Syk, Syk and GAPDH in neutrophils from Ceacam1^−/− mice reintroduced with empty vector (KO/(emp)), CEACAM1-2L (KO/(CC1-2L)), CEACAM1-4L (KO/(CC1-4L)), CEACAM1-2S (KO/(CC1-2S)), CEACAM1-4S (KO/(CC1-4S)), ITIMs mutated CEACAM1-2L (KO/(CC1-2 Lm)) and ITIMs mutated CEACAM1-4L (KO/(CC1-4 Lm)) with or without LPS treatment. (<b>D</b>) Immunoblot analysis of the p-Tyr, Syk, SHP-1 and CEACAM1 in neutrophils from KO/(emp), KO/(CC1-2L), KO/(CC1-4L), KO/(CC1-2S), KO/(CC1-4S), KO/(CC1-2 Lm) and KO/(CC1-4 Lm) chimeras after IP with CEACAM1 antibody. (<b>E</b>) IL-1β production in the supernatants of LPS treated neutrophils from KO/(emp), KO/(CC1-2L), KO/(CC1-4L), KO/(CC1-2S), KO/(CC1-4S), KO/(CC1-2 Lm) and KO/(CC1-4 Lm) chimeras. (<b>F</b>) Immunoblot analysis showing caspase-1 activation of LPS treated neutrophils KO/(emp), KO/(CC1-2L), KO/(CC1-4L), KO/(CC1-2S), KO/(CC1-4S), KO/(CC1-2 Lm) and KO/(CC1-4 Lm) chimeras. Data are representative of 3 different experiments and p values (<b>E</b>) were calculated by a 2-tailed T-test ***p≤0.001.</p