Lysosomal Cholesterol Hydrolysis Couples Efferocytosis to Anti-Inflammatory Oxysterol Production.

RATIONALE: Macrophages face a substantial amount of cholesterol after the ingestion of apoptotic cells, and the LIPA (lysosomal acid lipase) has a major role in hydrolyzing cholesteryl esters in the endocytic compartment. OBJECTIVE: Here, we directly investigated the role of LIPA-mediated clearance of apoptotic cells both in vitro and in vivo. METHODS AND RESULTS: We show that LIPA inhibition causes a defective efferocytic response because of impaired generation of 25-hydroxycholesterol and 27-hydroxycholesterol. Reduced synthesis of 25-hydroxycholesterol after LIPA inhibition contributed to defective mitochondria-associated membrane leading to mitochondrial oxidative stress-induced NLRP3 (NOD-like receptor family, pyrin domain containing) inflammasome activation and caspase-1-dependent Rac1 (Ras-related C3 botulinum toxin substrate 1) degradation. A secondary event consisting of failure to appropriately activate liver X receptor-mediated pathways led to mitigation of cholesterol efflux and apoptotic cell clearance. In mice, LIPA inhibition caused defective clearance of apoptotic lymphocytes and stressed erythrocytes by hepatic and splenic macrophages, culminating in splenomegaly and splenic iron accumulation under hypercholesterolemia. CONCLUSIONS: Our findings position lysosomal cholesterol hydrolysis as a critical process that prevents metabolic inflammation by enabling efficient macrophage apoptotic cell clearance.

Secreted IgM modulates IL-10 expression in B cells.

IL-10+ B cells are critical for immune homeostasis and restraining immune responses in infection, cancer, and inflammation; however, the signals that govern IL-10+ B cell differentiation are ill-defined. Here we find that IL-10+ B cells expand in mice lacking secreted IgM ((s)IgM-/-) up to 10-fold relative to wildtype (WT) among all major B cell and regulatory B cell subsets. The IL-10+ B cell increase is polyclonal and presents within 24 hours of birth. In WT mice, sIgM is produced prenatally and limits the expansion of IL-10+ B cells. Lack of the high affinity receptor for sIgM, FcµR, in B cells translates into an intermediate IL-10+ B cell phenotype relative to WT or sIgM-/- mice. Our study thus shows that sIgM regulates IL-10 programming in B cells in part via B cell-expressed FcµR, thereby revealing a function of sIgM in regulating immune homeostasis.

Type 1 diabetes associated and tissue transglutaminase autoantibodies in patients without type 1 diabetes and coeliac disease with confirmed viral infections.

Coeliac disease and type 1 diabetes are autoimmune diseases that may share the same initiating environmental factors. In this study, the occurrence of type 1 diabetes associated autoantibodies (GADA and IA-2A) and tissue transglutaminase autoantibodies (TGA) was determined in patients with confirmed viral infections and no signs of type 1 diabetes or coeliac disease. Serum samples from 82 Cuban patients tested positive for PCR and IgG specific to enterovirus (HEV, serotype echovirus 16, 20 samples), Epstein-Barr virus (EBV, 20 samples), cytomegalovirus (CMV, 21 samples), and hepatitis C virus (HCV, 21 samples); and sera from 164 controls negative serologically to EBV, CMV, HCV, and echovirus 16 were enrolled in the study. All subjects were screened for GADA, IA-2A, and TGA. The prevalence of TGA in patients infected with HEV, EBV, CMV, or HCV was 55% (11/20), 25% (5/20), 9.5% (2/21), and 9.5% (2/21), respectively. GADA and IA-2A were found in 15% (3/20) and 25% (5/20) of patients infected with HEV. None of the patients infected by EBV, CMV, and HCV had GADA or IA-2A. All children infected with HEV who were positive for type 1 diabetes-associated autoantibodies were also TGA-positive. None of the sera from uninfected subjects were positive for GADA, IA-2A or TGA. In conclusion, TGA can develop during infection with HEV, EBV, CMV, or HCV, while the emergence of islet cell related autoantibodies is restricted to HEV infections. The findings suggest that HEV may be a shared environmental factor for the development of islet and gut-related autoimmunity.

Skin-Homing Regulatory B Cells Required for Suppression of Cutaneous Inflammation.

Pro and anti-inflammatory B-cell subsets that localize to unperturbed and inflamed skin are newly emerging components of the skin immune system. To test the relevance of regulatory B cells (Bregs) in the suppression of cutaneous inflammation, we asked whether impaired migration of these cells into the skin exacerbates skin inflammation. Using a mouse model with a B-cell‒specific tamoxifen-inducible deletion of α4ß1 integrin, we demonstrate that selective disruption of α4ß1-integrin expression in B cells significantly decreases IL-10+ Bregs in inflamed skin, whereas it does not affect their counterparts in lymphoid tissues. Impaired skin homing and reduced cutaneous accumulation of IL-10+ Bregs lead to a significant increase in clinical and histopathological parameters of inflammation in both psoriasiform skin inflammation and cutaneous delayed contact hypersensitivity. Thus, our data show a crucial function of skin-homing IL-10+ Bregs in the suppression of skin inflammation, supporting the notion that Bregs are critical players in the cutaneous environment during inflammatory skin diseases.

B Cells and Melanoma Pathogenesis.

Melanoma is one of the deadliest cancers. In this issue, Kobayashi et al. (2019) demonstrate a novel role for tumor-infiltrating innate-like B1a B cells in promoting melanoma growth. IL-10+ (B1a) regulatory B cells accumulate selectively in melanomas, and enhance tumor growth through suppression of cytokine production by tumor-infiltrating CD8+ T cells and potentially additional IL-10-dependent mechanisms.

Caspase 1/11 Deficiency or Pharmacological Inhibition Mitigates Psoriasis-Like Phenotype in Mice.

Inflammatory caspases, activated within the inflammasome, are responsible for the maturation and secretion of IL-1ß/IL-18. Although their expression in psoriasis was shown several years ago, little is known about the role of inflammatory caspases in the context of psoriasis. Here, we confirmed that caspases 1, 4, and 5 are activated in lesional skin from psoriasis patients. We showed in three psoriasis-like models that inflammatory caspases are activated, and accordingly, caspase 1/11 invalidation or pharmacological inhibition by Ac-YVAD-CMK (i.e., Ac-Tyr-Val-Ala-Asp-chloromethylketone) injection induced a decrease in ear thickness, erythema, scaling, inflammatory cytokine expression, and immune cell infiltration in mice. We observed that keratinocytes were primed to secrete IL-1ß when cultured in conditions mimicking psoriasis. Generation of chimeric mice by bone marrow transplantation was carried out to decipher the respective contribution of keratinocytes and/or immune cells in the activation of inflammatory caspases during psoriasis-like inflammatory response. Our data showed that the presence of caspase 1/11 in the immune system is sufficient for a fully inflammatory response, whereas the absence of caspase 1/11 in keratinocytes/fibroblasts had no impact. In summary, our study indicates that inflammatory caspases activated in immune cells are implicated in psoriasis pathogenesis.

The oncogenic tyrosine kinase Lyn impairs the pro-apoptotic function of Bim.

Phosphorylation of Ser/Thr residues is a well-established modulating mechanism of the pro-apoptotic function of the BH3-only protein Bim. However, nothing is known about the putative tyrosine phosphorylation of this Bcl-2 family member and its potential impact on Bim function and subsequent Bax/Bak-mediated cytochrome c release and apoptosis. As we have previously shown that the tyrosine kinase Lyn could behave as an anti-apoptotic molecule, we investigated whether this Src family member could directly regulate the pro-apoptotic function of Bim. In the present study, we show that Bim is phosphorylated onto tyrosine residues 92 and 161 by Lyn, which results in an inhibition of its pro-apoptotic function. Mechanistically, we show that Lyn-dependent tyrosine phosphorylation of Bim increases its interaction with anti-apoptotic members such as Bcl-xL, therefore limiting mitochondrial outer membrane permeabilization and subsequent apoptosis. Collectively, our data uncover one molecular mechanism through which the oncogenic tyrosine kinase Lyn negatively regulates the mitochondrial apoptotic pathway, which may contribute to the transformation and/or the chemotherapeutic resistance of cancer cells.

Low-Protein Diet Induces IRE1α-Dependent Anticancer Immunosurveillance.

Dietary restriction (DR) was shown to impact on tumor growth with very variable effects depending on the cancer type. However, how DR limits cancer progression remains largely unknown. Here, we demonstrate that feeding mice a low-protein (Low PROT) isocaloric diet but not a low-carbohydrate (Low CHO) diet reduced tumor growth in three independent mouse cancer models. Surprisingly, this effect relies on anticancer immunosurveillance, as depleting CD8+ T cells, antigen-presenting cells (APCs), or using immunodeficient mice prevented the beneficial effect of the diet. Mechanistically, we established that a Low PROT diet induces the unfolded protein response (UPR) in tumor cells through the activation of IRE1α and RIG1 signaling, thereby resulting in cytokine production and mounting an efficient anticancer immune response. Collectively, our data suggest that a Low PROT diet induces an IRE1α-dependent UPR in cancer cells, enhancing a CD8-mediated T cell response against tumors.

Safety and Immunogenicity of a Human Epidermal Growth Factor Receptor 1 (HER1)-Based Vaccine in Prostate Castration-Resistant Carcinoma Patients: A Dose-Escalation Phase I Study Trial.

Metastatic castration-resistant prostate cancer (CRPC) remains incurable due to the lack of effective therapies. Activation of the human epidermal growth factor receptor 1 (HER1) in prostate cancer contributes to metastatic progression as well as to disease relapse. Here, we determined the toxicity and immunogenicity of a HER1-based cancer vaccine in CRPC patients included in a phase I clinical trial. CRPC patients (n = 24) were intramuscularly vaccinated with HER1 vaccine consisting of the extracellular domain of HER1 molecule (ECD) and very small size proteoliposome from Neisseria meningitidis (VSSP) and Montanide ISA-51 VG as adjuvants. Patients were included in five groups according to the vaccine dose (100, 200, 400, 600, and 800 µg). The primary endpoints were safety and immunogenicity. The anti-HER1 antibodies were measured by an ELISA, the recognition of an HER1 positive tumor cell line and the inhibition of HER1 phosphorylation by sera were determined by flow cytometry and western blot analysis, respectively. The HER1-specific T cell response was assessed by determination of IFN-γ-producing T cells using ELISpot assay. The vaccine was well tolerated. No grade III or IV adverse events were reported. High titers of anti-HER1 antibodies were observed in most of the evaluated patients. There were no significant differences regarding the geometric means of the anti-HER1 titers among the dose groups except the group of 100 µg in which antibody titers were significantly lower. A Th1-type IgG subclasses pattern was predominant in most patients. Only patients receiving the higher doses of vaccine showed significant tumor cell recognition and HER1 phosphorylation inhibition by hyperimmune sera. Forty two percent of the patients showed a specific T cell response against HER1 peptides pool in post-treatment samples. There was a trend toward survival benefit in those patients showing high anti-HER1 specific antibody titers and a significant association between cellular immune response and clinical outcome.

Therapeutic Targeting of CD6 in Autoimmune Diseases: A Review of Cuban Clinical Studies with the Antibodies IOR-T1 and Itolizumab.

The CD6 molecule is a pan T cell marker involved in T cell regulation. Although CD6 expression has been correlated with human autoimmune diseases, only a few therapeutic approaches are exploring this molecule as target in the clinic. The biological functions and mechanisms of actions of CD6 have not been definitively established. It is probable that this molecule plays a dual role as a modulator of intracellular signaling. Itolizumab is a humanized monoclonal antibody specific for human CD6, developed at the Center of Molecular Immunology in Havana, Cuba. Its parent murine antibody, the IOR-T1 mAb, had been obtained in the 80's at the Institute of Oncology and Radiology, also in Havana. This article provides an overview of the clinical data obtained in Cuban patients with autoimmune diseases who have been treated with IOR-T1 mAb or itolizumab. Furthermore, we discuss the possible mechanism of action of itolizumab basing the analysis on recent site mutagenesis and structural data, which, contrary to previous interpretations, points to a steric blocking of the CD6-CD166 interaction in the cellular context. Overall, the conducted clinical studies have demonstrated that itolizumab has favorable clinical effects and a safety profile when used as monotherapy in patients with rheumatoid arthritis and psoriasis. So far, in vitro and in vivo evidences indicate that itolizumab has immunomodulatory and anti-inflammatory effects. Hence, itolizumab represents a new therapeutic option for autoimmune diseases such as rheumatoid arthritis and psoriasis.

Immunological evaluation of rheumatoid arthritis patients treated with itolizumab.

Rheumatoid arthritis is an autoimmune disease characterized by joint inflammation that affects approximately 1% of the general population. Itolizumab, a monoclonal antibody specific for the human CD6 molecule mainly expressed on T lymphocytes, has been shown to inhibit proliferation of T cells and proinflammatory cytokine production in psoriasis patients. We have now assessed the immunological effect of itolizumab in combination with methotrexate in rheumatoid arthritis by analyzing clinical samples taken from 30 patients enrolled in a clinical trial. T and B cell subpopulations were measured at different time points of the study. Plasma cytokine levels and anti-idiotypic antibody response to itolizumab were also evaluated. The combined treatment of itolizumab and methotrexate led to a reduction in the frequency of T cell subpopulations, and plasma levels of proinflammatory cytokines showed a significant decrease up to at least 12 weeks after treatment ended. No anti-idiotypic antibody response was detected. These results support the relevance of the CD6 molecule as a therapeutic target for the treatment of this disease.

Immunological and histological evaluation of clinical samples from psoriasis patients treated with anti-CD6 itolizumab.

Psoriasis is a chronic inflammatory disease with a prevalence of approximately 2-3% in the general population. The majority of diagnosed patients have plaque psoriasis, and about 20% have moderate-to-severe disease. Itolizumab, a new monoclonal antibody specific for the CD6 molecule mainly expressed on T lymphocytes, has demonstrated to inhibit in vitro ligand-induced proliferation and pro-inflammatory cytokine production. We assessed the immunological and histopathological effect of the antibody using clinical samples taken from 26 patients with moderate-to-severe psoriasis included in a clinical trial. The precursor frequency of lymphocytes activated with anti-CD2/CD3/CD28 beads, as well as the number of interferon (IFN)-γ-secreting T cells after stimulation, were measured at different time points of the study. Serum cytokine levels and anti-idiotypic antibody response to itolizumab were also evaluated. Additionally, lymphocyte infiltration and epidermis hyperplasia were studied in five patients. A significant reduction in T cell proliferation capacity and number of IFN-γ-producing T cells was found in treated patients. Serum levels of interleukin-6, tumor necrosis factor and IFN-γ showed an overall trend toward reduction. No anti-idiotypic antibody response was detected. A significant reduction in the epidermis hyperplasia was observed in analyzed patients. These results support the relevance of the CD6 molecule as a therapeutic target for the treatment of this disease.