The chemerin/CMKLR1 axis regulates intestinal graft-versus-host disease.

Gastrointestinal graft-versus-host disease (GvHD) is a major cause of mortality and morbidity following allogeneic bone marrow transplantation (allo-BMT). Chemerin is a chemotactic protein that recruits leukocytes to inflamed tissues by interacting with ChemR23/CMKLR1, a chemotactic receptor expressed by leukocytes, including macrophages. During acute GvHD, chemerin plasma levels were strongly increased in allo-BM-transplanted mice. The role of the chemerin/CMKLR1 axis in GvHD was investigated using Cmklr1-KO mice. WT mice transplanted with an allogeneic graft from Cmklr1-KO donors (t-KO) had worse survival and more severe GvHD. Histological analysis demonstrated that the gastrointestinal tract was the organ mostly affected by GvHD in t-KO mice. The severe colitis of t-KO mice was characterized by massive neutrophil infiltration and tissue damage associated with bacterial translocation and exacerbated inflammation. Similarly, Cmklr1-KO recipient mice showed increased intestinal pathology in both allogeneic transplant and dextran sulfate sodium-induced colitis. Notably, the adoptive transfer of WT monocytes into t-KO mice mitigated GvHD manifestations by decreasing gut inflammation and T cell activation. In patients, higher chemerin serum levels were predictive of GvHD development. Overall, these results suggest that CMKLR1/chemerin may be a protective pathway for the control of intestinal inflammation and tissue damage in GvHD.

Leukocyte cell-derived chemotaxin 2 is an antiviral regulator acting through the proto-oncogene MET.

Retinoic acid-inducible gene (RIG)-I is an essential innate immune sensor that recognises pathogen RNAs and induces interferon (IFN) production. However, little is known about how host proteins regulate RIG-I activation. Here, we show that leukocyte cell-derived chemotaxin 2 (LECT2), a hepatokine and ligand of the MET receptor tyrosine kinase is an antiviral regulator that promotes the RIG-I-mediated innate immune response. Upon binding to MET, LECT2 induces the recruitment of the phosphatase PTP4A1 to MET and facilitates the dissociation and dephosphorylation of phosphorylated SHP2 from MET, thereby protecting RIG-I from SHP2/c-Cbl-mediated degradation. In vivo, LECT2 overexpression enhances RIG-I-dependent IFN production and inhibits lymphocytic choriomeningitis virus (LCMV) replication in the liver, whereas these changes are reversed in LECT2 knockout mice. Forced suppression of MET abolishes IFN production and antiviral activity in vitro and in vivo. Interestingly, hepatocyte growth factor (HGF), an original MET ligand, inhibits LECT2-mediated anti-viral signalling; conversely, LECT2-MET signalling competes with HGF-MET signalling. Our findings reveal previously unrecognized crosstalk between MET-mediated proliferation and innate immunity and suggest that targeting LECT2 may have therapeutic value in infectious diseases and cancer.

Caspase-1 and interleukin-18 in children with post infectious bronchiolitis obliterans: a case-control study.

The exact immunological mechanisms of post infectious bronchiolitis obliterans (PIBO) in childhood are not fully known. It has been shown that the inflammasome and IL-18 pathway play important roles in the pathogenesis of lung fibrosis. We aimed to investigate the role of caspase-1, IL-18, and IL-18 components in PIBO. From January to May 2020, children with PIBO, children with history of influenza infection without PIBO, and healthy children were asked to participate in the study in three pediatric pulmonology centers. Serum caspase-1, IL-18, IL-18BP, IL-18R, and INF-γ levels were measured by ELISA and compared between the 3 groups. There were 21 children in the PIBO group, 16 children in the influenza group, and 39 children in the healthy control group. No differences in terms of age and gender between the 3 groups were found. IL-18 and IL-18BP levels were higher in the healthy control group (p = 0.018, p = 0.005, respectively). IL-18R was higher in the PIBO group (p = 0.001) and caspase-1 was higher in the PIBO and influenza group than the healthy control group (p = 0.002). IFN-γ levels did not differ between the 3 groups. IL-18BP/IL-18 was higher in the influenza group than the PIBO group and the healthy control group (p = 0.003). CONCLUSIONS: Caspase-1 level was increased in patients with PIBO which suggests that inflammasome activation may have a role in fibrosis; however, IL-18 level was found to be low. Mediators other than IL-18 may be involved in the inflammatory pathway in PIBO. Further immunological studies investigating inflammasome pathway are needed for PIBO with chronic inflammation. WHAT IS KNOWN: • Post infectious bronchiolitis obliterans (PIBO) is a rare, severe chronic lung disease during childhood which is associated with inflammation and fibrosis which lead to partial or complete luminal obstruction especially in small airways. • The exact immunological mechanisms of PIBO in childhood are not fully known. WHAT IS NEW: • Inflammasome activation persists even years after acute infection and may play a role in fibrosis in PIBO. • Mediators other than IL-18 may be involved in these inflammatory pathway.

Human-Induced CD49a+ NK Cells Promote Fetal Growth.

CD49a+ natural killer (NK) cells play a critical role in promoting fetal development and maintaining immune tolerance at the maternal-fetal interface during the early stages of pregnancy. However, given their residency in human tissue, thorough studies and clinical applications are difficult to perform. It is still unclear as to how functional human CD49a+ NK cells can be induced to benefit pregnancy outcomes. In this study, we established three no-feeder cell induction systems to induce human CD49a+ NK cells from umbilical cord blood hematopoietic stem cells (HSCs), bone marrow HSCs, and peripheral blood NK cells in vitro. These induced NK cells (iNKs) from three cell induction systems display high levels of CD49a, CD9, CD39, CD151 expression, low levels of CD16 expression, and no obvious cytotoxic capability. They are phenotypically and functionally similar to decidual NK cells. Furthermore, these iNKs display a high expression of growth-promoting factors and proangiogenic factors and can promote fetal growth and improve uterine artery blood flow in a murine pregnancy model in vivo. This research demonstrates the ability of human-induced CD49a+ NK cells to promote fetal growth via three cell induction systems, which could eventually be used to treat patients experiencing adverse pregnancy outcomes.

The Postnatal Leptin Surge Supports Immune Cell Function in Rats.

BACKGROUND: Leptin plays an important role in the regulation of the immune response. There is a physiological surge of leptin in rodents during the neonatal period, which has mainly been studied in the context of brain development. However, little is known about the effects of this neonatal leptin surge on immunity. Therefore, we investigated whether blocking this leptin surge could affect several immune functions. METHODS: Male and female rats were injected subcutaneously with 5 mg/Kg/day of rat pegylated super leptin antagonist during the neonatal period (PND5-9). On the peripubertal period, relevant functions as well as cytokine release by spleen leukocytes were studied in these animals. RESULTS: The results showed that the animals significantly display an impaired anti-tumor NK activity and chemotactic and proliferation capacity of lymphocytes in response to mitogens. In addition, several cytokine concentrations, released under mitogen-stimulated conditions, were also altered. CONCLUSION: In conclusion, the neonatal leptin surge seems to be involved in the establishment of an adequate immune response and cytokine profile, which are crucial for the maintenance of a healthy life.

Immunological Aspects of AXL/GAS-6 in the Context of Human Liver Regeneration.

AXL and its corresponding ligand growth arrest-specific 6 (GAS-6) are critically involved in hepatic immunomodulation and regenerative processes. Pleiotropic inhibitory effects on innate inflammatory responses might essentially involve the shift of macrophage phenotype from a pro-inflammatory M1 to an anti-inflammatory M2. We aimed to assess the relevance of the AXL/GAS-6-pathway in human liver regeneration and, consequently, its association with clinical outcome after hepatic resection. Soluble AXL (sAXL) and GAS-6 levels were analyzed at preoperative and postoperative stages in 154 patients undergoing partial hepatectomy and correlated with clinical outcome. Perioperative dynamics of interleukin (IL)-6, soluble tyrosine-protein kinase MER (sMerTK), soluble CD163 (sCD163), and cytokeratin (CK) 18 were assessed to reflect pathophysiological processes. Preoperatively elevated sAXL and GAS-6 levels predicted postoperative liver dysfunction (area under the curve = 0.721 and 0.722; P < 0.005) and worse clinical outcome. These patients failed to respond with an immediate increase of sAXL and GAS-6 upon induction of liver regeneration. Abolished AXL pathway response resulted in a restricted increase of sCD163, suggesting a disrupted phenotypical switch to regeneratory M2 macrophages. No association with sMerTK was observed. Concomitantly, a distinct association of IL-6 levels with an absent increase of AXL/GAS-6 signaling indicated pronounced postoperative inflammation. This was further supported by increased intrahepatic secondary necrosis as reflected by CK18M65. sAXL and GAS-6 represent not only potent and easily accessible preoperative biomarkers for the postoperative outcome but also AXL/GAS-6 signaling might be of critical relevance in human liver regeneration. Refractory AXL/GAS-6 signaling, due to chronic overactivation/stimulation in the context of underlying liver disease, appears to abolish their immediate release following induction of liver regeneration, causing overwhelming immune activation, presumably via intrahepatic immune regulation.

Breastfeeding and circulating immunological markers during the first 3 years of life: the DIABIMMUNE study.

AIMS/HYPOTHESIS: Our aim was to study the association between duration of breastfeeding and circulating immunological markers during the first 3 years of life in children with HLA-conferred susceptibility to type 1 diabetes. METHODS: We performed a longitudinal analysis of 38 circulating immunological markers (cytokines, chemokines and growth factors) in serum samples from Finnish (56 individuals, 147 samples), Estonian (56 individuals 148 samples) and Russian Karelian children (62 individuals, 149 samples) at 3, 6, 12, 18, 24 and 36 months of age. We also analysed gut inflammation markers (calprotectin and human ß defensin-2) at 3 (n = 96) and 6 months (n = 153) of age. Comparisons of immunological marker medians were performed between children who were breastfed for 6 months or longer vs children who were breastfed for less than 6 months. RESULTS: Breastfeeding for 6 months or longer vs less than 6 months was associated with lower median of serum immunological markers at 6 months (granulocyte-macrophage colony-stimulating factor [GMCSF], macrophage inflammatory protein [MIP-3α]), 12 months (IFN-α2, vascular endothelial growth factor, GMCSF, IFN-γ, IL-21), 18 months (FGF-2, IFN-α2) and 24 months of age (CCL11 [eotaxin], monocyte chemoattractant protein-1, TGFα, soluble CD40 ligand, IL-13, IL-21, IL-5, MIP-1α) (all p < 0.01) but not at 36 months of age. Breastfeeding was not associated with gut inflammation markers at 3 and 6 months of age. CONCLUSIONS/INTERPRETATION: Children who were breastfed for 6 months or longer had lower medians for 14 immunological markers at one or more age points during the first 2 years of life compared with children who were breastfed for less than 6 months. The clinical meaning of the findings is not clear. However, the present study contributes to the understanding of immunological differences in children that have been breastfed longer, and thus provides a mechanistic suggestion for the previously observed associations between breastfeeding and risk of type 1 diabetes.

BPIFB4 Circulating Levels and Its Prognostic Relevance in COVID-19.

Aging and comorbidities make individuals at greatest risk of COVID-19 serious illness and mortality due to senescence-related events and deleterious inflammation. Long-living individuals (LLIs) are less susceptible to inflammation and develop more resiliency to COVID-19. As demonstrated, LLIs are characterized by high circulating levels of BPIFB4, a protein involved in homeostatic response to inflammatory stimuli. Also, LLIs show enrichment of homozygous genotype for the minor alleles of a 4 missense single-nucleotide polymorphism haplotype (longevity-associated variant [LAV]) in BPIFB4, able to counteract progression of diseases in animal models. Thus, the present study was designed to assess the presence and significance of BPIFB4 level in COVID-19 patients and the potential therapeutic use of LAV-BPIFB4 in fighting COVID-19. BPIFB4 plasma concentration was found significantly higher in LLIs compared to old healthy controls while it significantly decreased in 64 COVID-19 patients. Further, the drop in BPIFB4 values correlated with disease severity. Accordingly to the LAV-BPIFB4 immunomodulatory role, while lysates of SARS-CoV-2-infected cells induced an inflammatory response in healthy peripheral blood mononuclear cells in vitro, the co-treatment with recombinant protein (rh) LAV-BPIFB4 resulted in a protective and self-limiting reaction, culminating in the downregulation of CD69 activating-marker for T cells (both TCD4+ and TCD8+) and in MCP-1 reduction. On the contrary, rhLAV-BPIFB4 induced a rapid increase in IL-18 and IL-1b levels, shown largely protective during the early stages of the virus infection. This evidence, along with the ability of rhLAV-BPIFB4 to counteract the cytotoxicity induced by SARS-CoV-2 lysate in selected target cell lines, corroborates BPIFB4 prognostic value and open new therapeutic possibilities in more vulnerable people.

The Bigger Picture: Why Oral Mucosa Heals Better Than Skin.

Wound healing is an essential process to restore tissue integrity after trauma. Large skin wounds such as burns often heal with hypertrophic scarring and contractures, resulting in disfigurements and reduced joint mobility. Such adverse healing outcomes are less common in the oral mucosa, which generally heals faster compared to skin. Several studies have identified differences between oral and skin wound healing. Most of these studies however focus only on a single stage of wound healing or a single cell type. The aim of this review is to provide an extensive overview of wound healing in skin versus oral mucosa during all stages of wound healing and including all cell types and molecules involved in the process and also taking into account environmental specific factors such as exposure to saliva and the microbiome. Next to intrinsic properties of resident cells and differential expression of cytokines and growth factors, multiple external factors have been identified that contribute to oral wound healing. It can be concluded that faster wound closure, the presence of saliva, a more rapid immune response, and increased extracellular matrix remodeling all contribute to the superior wound healing and reduced scar formation in oral mucosa, compared to skin.

Neutrophil in the Pancreatic Tumor Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is a malignancy with a poor prognosis and low survival rates. PDAC is characterized by a fibroinflammatory tumor microenvironment enriched by abundant fibroblasts and a variety of immune cells, contributing to its aggressiveness. Neutrophils are essential infiltrating immune cells in the PDAC microenvironment. Recent studies have identified several cellular mechanisms by which neutrophils are recruited to tumor lesion and promote tumorigenesis. This review summarizes the current understanding of the interplay between neutrophils, tumor cells, and other components in the PDAC tumor microenvironment. The prognosis and therapeutic implications of neutrophils in PDAC are also discussed.

The chemerin-CMKLR1 axis limits thermogenesis by controlling a beige adipocyte/IL-33/type 2 innate immunity circuit.

IL-33-associated type 2 innate immunity has been shown to support beige fat formation and thermogenesis in subcutaneous inguinal white adipose tissue (iWAT), but little is known about how it is regulated in iWAT. Chemerin, as a newly identified adipokine, is clinically associated with obesity and metabolic disorders. We here show that cold exposure specifically reduces chemerin and its receptor chemerin chemokine-like receptor 1 (CMKLR1) expression in iWAT. Lack of chemerin or adipocytic CMKLR1 enhances cold-induced thermogenic beige fat via potentiating type 2 innate immune responses. Mechanistically, we identify adipocytes, particularly beige adipocytes, as the main source for cold-induced IL-33, which is restricted by the chemerin-CMKLR1 axis via dampening cAMP-PKA signaling, thereby interrupting a feed-forward circuit between beige adipocytes and type 2 innate immunity that is required for cold-induced beige fat and thermogenesis. Moreover, specific deletion of adipocytic IL-33 inhibits cold-induced beige fat and type 2 innate immune responses. Last, genetic blockade of adipocytic CMKLR1 protects against diet-induced obesity and enhances the metabolic benefits of cold stimulation in preestablished obese mice. Thus, our study identifies the chemerin-CMKLR1 axis as a physiological negative regulator of thermogenic beige fat via interrupting adipose-immune communication and suggests targeting adipose CMKLR1 as a potential therapeutic strategy for obesity-related metabolic disorders.

Trichuris muris infection drives cell-intrinsic IL4R alpha independent colonic RELMα+ macrophages.

The intestinal nematode parasite Trichuris muris dwells in the caecum and proximal colon driving an acute resolving intestinal inflammation dominated by the presence of macrophages. Notably, these macrophages are characterised by their expression of RELMα during the resolution phase of the infection. The RELMα+ macrophage phenotype associates with the presence of alternatively activated macrophages and work in other model systems has demonstrated that the balance of classically and alternatively activated macrophages is critically important in enabling the resolution of inflammation. Moreover, in the context of type 2 immunity, RELMα+ alternatively activated macrophages are associated with the activation of macrophages via the IL4Rα. Despite a breadth of inflammatory pathologies associated with the large intestine, including those that accompany parasitic infection, it is not known how colonic macrophages are activated towards an alternatively activated phenotype. Here, we address this important knowledge gap by using Trichuris muris infection, in combination with transgenic mice (IL4Rαfl/fl.CX3CR1Cre) and IL4Rα-deficient/wild-type mixed bone marrow chimaeras. We make the unexpected finding that education of colonic macrophages towards a RELMα+, alternatively activated macrophage phenotype during T. muris infection does not require IL4Rα expression on macrophages. Further, this independence is maintained even when the mice are treated with an anti-IFNγ antibody during infection to create a strongly polarised Th2 environment. In contrast to RELMα, PD-L2 expression on macrophages post infection was dependent on IL4Rα signalling in the macrophages. These novel data sets are important, revealing a surprising cell-intrinsic IL4R alpha independence of the colonic RELMα+ alternatively activated macrophage during Trichuris muris infection.

SLIT2/ROBO signaling in tumor-associated microglia and macrophages drives glioblastoma immunosuppression and vascular dysmorphia.

SLIT2 is a secreted polypeptide that guides migration of cells expressing Roundabout 1 and 2 (ROBO1 and ROBO2) receptors. Herein, we investigated SLIT2/ROBO signaling effects in gliomas. In patients with glioblastoma (GBM), SLIT2 expression increased with malignant progression and correlated with poor survival and immunosuppression. Knockdown of SLIT2 in mouse glioma cells and patient-derived GBM xenografts reduced tumor growth and rendered tumors sensitive to immunotherapy. Tumor cell SLIT2 knockdown inhibited macrophage invasion and promoted a cytotoxic gene expression profile, which improved tumor vessel function and enhanced efficacy of chemotherapy and immunotherapy. Mechanistically, SLIT2 promoted microglia/macrophage chemotaxis and tumor-supportive polarization via ROBO1- and ROBO2-mediated PI3K-γ activation. Macrophage Robo1 and Robo2 deletion and systemic SLIT2 trap delivery mimicked SLIT2 knockdown effects on tumor growth and the tumor microenvironment (TME), revealing SLIT2 signaling through macrophage ROBOs as a potentially novel regulator of the GBM microenvironment and immunotherapeutic target for brain tumors.

Mediterranean fever gene variants modify clinical phenotypes of idiopathic multi-centric Castleman disease.

Four cases of idiopathic multi-centric Castleman disease (iMCD) reportedly have variants in hereditary autoinflammatory disease-related genes; however, the frequency and role of these variants in iMCD is still unknown. We therefore investigated such gene variants among patients with iMCD and aimed to reveal the relationship between iMCD and autoinflammatory disease-related genes. We reviewed 14 Japanese iMCD patients who were recruited between January 2015 and September 2019. All patients met both the Japanese tentative diagnostic criteria for Castleman disease and the international consensus diagnostic criteria for iMCD. We performed genetic analyses for 31 autoinflammatory disease-related genes by targeted next-generation sequencing. The MEFV gene variants were observed in 10 of 14 patients with iMCD. Although iMCD had a high percentage of exons 2 or 3 variants of MEFV, comparison of data from healthy Japanese subjects indicated that there was no significant difference in the percentage between healthy Japanese subjects and patients with iMCD. Variants of uncertain significance (VUS) in the TNFRSF1A and CECR1 genes were observed in two of the patients, respectively. We divided patients into two groups-those with MEFV variants (excluding E148Q variants) and those without MEFV variants-and compared the clinical characteristics between these two groups. Patients with MEFV variants, excluding E148Q variants, exhibited a significantly higher likelihood of fever and significantly lower levels of hemoglobin than those lacking MEFV variants. Our results indicated that patients with iMCD tended to have a high frequency of MEFV gene variants and the presence of such variants can affect iMCD clinical phenotypes.

The Role of Hypoxia-Induced Mitogenic Factor in Organ-Specific Inflammation in the Lung and Liver: Key Concepts and Gaps in Knowledge Regarding Molecular Mechanisms of Acute or Immune-Mediated Liver Injury.

Hypoxia-induced mitogenic factor (HIMF), which is also known as resistin-like molecule α (RELM-α), found in inflammatory zone 1 (FIZZ1), or resistin-like alpha (retlna), is a cysteine-rich secretory protein and cytokine. HIMF has been investigated in the lung as a mediator of pulmonary fibrosis, inflammation and as a marker for alternatively activated macrophages. Although these macrophages have been found to have a role in acute liver injury and acetaminophen toxicity, few studies have investigated the role of HIMF in acute or immune-mediated liver injury. The aim of this focused review is to analyze the literature and examine the effects of HIMF and its human homolog in organ-specific inflammation in the lung and liver. We followed the guidelines set by PRISMA in constructing this review. The relevant checklist items from PRISMA were included. Items related to meta-analysis were excluded because there were no randomized controlled clinical trials. We found that HIMF was increased in most models of acute liver injury and reduced damage from acetaminophen-induced liver injury. We also found strong evidence for HIMF as a marker for alternatively activated macrophages. Our overall risk of bias assessment of all studies included revealed that 80% of manuscripts demonstrated some concerns in the randomization process. We also demonstrated some concerns (54.1%) and high risk (45.9%) of bias in the selection of the reported results. The need for randomization and reduction of bias in the reported results was similarly detected in the studies that focused on HIMF and the liver. In conclusion, we propose that HIMF could be utilized as a marker for M2 macrophages in immune-mediated liver injury. However, we also detected the need for randomized clinical trials and additional experimental and human prospective studies in order to fully comprehend the role of HIMF in acute or immune-mediated liver injury.

Genetic Variability of Human Cytomegalovirus Clinical Isolates Correlates With Altered Expression of Natural Killer Cell-Activating Ligands and IFN-γ.

Human cytomegalovirus (HCMV) infection often leads to systemic disease in immunodeficient patients and congenitally infected children. Despite its clinical significance, the exact mechanisms contributing to HCMV pathogenesis and clinical outcomes have yet to be determined. One of such mechanisms involves HCMV-mediated NK cell immune response, which favors viral immune evasion by hindering NK cell-mediated cytolysis. This process appears to be dependent on the extent of HCMV genetic variation as high levels of variability in viral genes involved in immune escape have an impact on viral pathogenesis. However, the link between viral genome variations and their functional effects has so far remained elusive. Thus, here we sought to determine whether inter-host genetic variability of HCMV influences its ability to modulate NK cell responses to infection. For this purpose, five HCMV clinical isolates from a previously characterized cohort of pediatric patients with confirmed HCMV congenital infection were evaluated by next-generation sequencing (NGS) for genetic polymorphisms, phylogenetic relationships, and multiple-strain infection. We report variable levels of genetic characteristics among the selected clinical strains, with moderate variations in genome regions associated with modulation of NK cell functions. Remarkably, we show that different HCMV clinical strains differentially modulate the expression of several ligands for the NK cell-activating receptors NKG2D, DNAM-1/CD226, and NKp30. Specifically, the DNAM-1/CD226 ligand PVR/CD155 appears to be predominantly upregulated by fast-replicating ("aggressive") HCMV isolates. On the other hand, the NGK2D ligands ULBP2/5/6 are downregulated regardless of the strain used, while other NK cell ligands (i.e., MICA, MICB, ULBP3, Nectin-2/CD112, and B7-H6) are not significantly modulated. Furthermore, we show that IFN-γ; production by NK cells co-cultured with HCMV-infected fibroblasts is directly proportional to the aggressiveness of the HCMV clinical isolates employed. Interestingly, loss of NK cell-modulating genes directed against NK cell ligands appears to be a common feature among the "aggressive" HCMV strains, which also share several gene variants across their genomes. Overall, even though further studies based on a higher number of patients would offer a more definitive scenario, our findings provide novel mechanistic insights into the impact of HCMV genetic variability on NK cell-mediated immune responses.

Expansion of an Unusual Virtual Memory CD8+ Subpopulation Bearing Vα3.2 TCR in Themis-Deficient Mice.

Deletion of the gene for Themis affects T cell selection in the thymus, which would be expected to affect the TCR repertoire. We found an increased proportion of cells expressing Vα3.2 (TRAV9N-3) in the peripheral CD8+ T cell population in mice with germline Themis deficiency. Analysis of the TCRα repertoire indicated it was generally reduced in diversity in the absence of Themis, whereas the diversity of sequences using the TRAV9N-3 V-region element was increased. In wild type mice, Vα3.2+ cells showed higher CD5, CD6 and CD44 expression than non-Vα3-expressing cells, and this was more marked in cells from Themis-deficient mice. This suggested a virtual memory phenotype, as well as a stronger response to self-pMHC. The Vα3.2+ cells responded more strongly to IL-15, as well as showing bystander effector capability in a Listeria infection. Thus, the unusually large population of Vα3.2+ CD8+ T cells found in the periphery of Themis-deficient mice reflects not only altered thymic selection, but also allowed identification of a subset of bystander-competent cells that are also present in wild-type mice.

The role of CECR1 in the immune-modulatory effects of butyrate and correlation between ADA2 and M1/M2 chemokines in tuberculous pleural effusion.

OBJECTIVES: The Cat Eye Syndrome Critical Region, Candidate 1 (CECR1) gene encoding adenosine deaminase 2 (ADA2) is mainly expressed by macrophages. Given the immunomodulatory functions of butyrate, we examined the effect of butyrate on CECR1 expression of macrophages and the relationship between ADA2 and M1/M2 macrophages-associated chemokines in pleural fluid of patients with tuberculous pleural effusion (TPE). METHODS: Expression of CECR1 was evaluated in lipopolysaccharide (LPS)-stimulated and/or butyrate treated THP-1 cells. The role of CECR1 on butyrate-induced immune response was evaluated using siRNA transfected THP-1 cells. M1/M2 chemokines and ADA2 were measured in pleural fluid of patients with TPE. RESULTS: Butyrate promoted the expression of CECR1 and M2-macrophage markers in THP-1 cells. CECR1 was found to be involved in regulating M2 polarization in THP-1 cells treated with LPS and butyrate. Among chemokines measured in pleural fluid of patients with TPE, there was a significant negative correlation between CCL21 and ADA2 levels and between CCL25 and ADA2 levels, and a significant positive correlation between TGF-ß and ADA2 levels and between IL-22 and ADA2 levels. CONCLUSIONS: CECR1 played an important role in the butyrate-modulated inflammatory responses in LPS-stimulated THP-1 cells. ADA2 may exert anti-inflammatory effects during the process of pleural inflammation in patients with TPE.

Platelet Lysate Nebulization Protocol for the Treatment of COVID-19 and Its Sequels: Proof of Concept and Scientific Rationale.

One of the most severe effects of coronavirus disease 2019 (COVID-19) is lung disorders such as acute respiratory distress syndrome. In the absence of effective treatments, it is necessary to search for new therapies and therapeutic targets. Platelets play a fundamental role in respiratory disorders resulting from viral infections, being the first line of defense against viruses and essential in maintaining lung function. The direct application of platelet lysate (PL) obtained from the platelet-rich plasma of healthy donors could help in the improvement of the patient due its anti-inflammatory, immunomodulatory, antifibrotic, and repairing effects. This work evaluates PL nebulization by analyzing its levels of growth factors and its biological activity on lung fibroblast cell cultures, besides describing a scientific basis for its use in this kind of pathology. The data of the work suggest that the molecular levels and biological activity of the PL are maintained after nebulization. Airway administration would allow acting directly on the lung tissue modulating inflammation and stimulating reparative processes on key structures such as the alveolocapillary barrier, improving the disease and sequels. The protocol developed in this work is a first step for the study of nebulized PL both in animal experimentation and in clinical trials.

A Novel Artificially Humanized Anti-Cripto-1 Antibody Suppressing Cancer Cell Growth.

Cripto-1 is a member of the EGF-CFC/FRL1/Cryptic family and is involved in embryonic development and carcinogenesis. We designed a novel anti-Cripto-1 artificial antibody and assessed the recognition to the antigen and the potential to suppress the growth of cancer stem cells. First, single chain antibody clones were isolated by bio-panning with the affinity to recombinant Cripto-1 protein from our original phage-display library. Then, the variable regions of heavy chain VH and light chain VL in each clone were fused to constant regions of heavy chain CH and light chain CL regions respectively. These fused genes were expressed in ExpiCHO-S cells to produce artificial humanized antibodies against Cripto-1. After evaluation of the expression levels, one clone was selected and the anti-Cripto-1 antibody was produced and purified. The purified antibody showed affinity to recombinant Cripto-1 at 1.1 pmol and immunoreactivity to cancer tissues and cell lines. The antibody was available to detect the immunoreactivity in tissue microarrays of malignant tumors as well as in Cripto-1 overexpressing cells. Simultaneously, the antibody exhibited the potential to suppress the growth of human colon cancer derived GEO cells overexpressing Cripto-1 with IC50 at approximately 110 nM. The artificially humanized antibody is proposed to be a good candidate to target cancer cells overexpressing Cripto-1.