Neutralizing antibodies with neurotropic factor treatment maintain neurodevelopmental gene expression upon exposure to human cytomegalovirus.

IMPORTANCE: Human cytomegalovirus (HCMV) infection is the leading cause of non-heritable birth defects worldwide. HCMV readily infects the early progenitor cell population of the developing brain, and we have found that infection leads to significantly downregulated expression of key neurodevelopmental transcripts. Currently, there are no approved therapies to prevent or mitigate the effects of congenital HCMV infection. Therefore, we used human-induced pluripotent stem cell-derived organoids and neural progenitor cells to elucidate the glycoproteins and receptors used in the viral entry process and whether antibody neutralization was sufficient to block viral entry and prevent disruption of neurodevelopmental gene expression. We found that blocking viral entry alone was insufficient to maintain the expression of key neurodevelopmental genes, but neutralization combined with neurotrophic factor treatment provided robust protection. Together, these studies offer novel insight into mechanisms of HCMV infection in neural tissues, which may aid future therapeutic development.

Lymph-Node-Based CD3+ CD20+ Cells Emerge from Membrane Exchange between T Follicular Helper Cells and B Cells and Increase Their Frequency following Simian Immunodeficiency Virus Infection.

CD4+ T follicular helper (TFH) cells are key targets for human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) replication and contribute to the virus reservoir under antiretroviral therapy (ART). Here, we describe a novel CD3+ CD20+ double-positive (DP) lymphocyte subset, resident in secondary lymphoid organs of humans and rhesus macaques (RMs), that appear predominantly after membrane exchange between TFH and B cells. DP lymphocytes are enriched in cells displaying a TFH phenotype (CD4+ PD1hi CXCR5hi), function (interleukin 21 positive [IL-21+]), and gene expression profile. Importantly, expression of CD40L upon brief in vitro mitogen stimulation identifies, by specific gene-expression signatures, DP cells of TFH-cell origin versus those of B-cell origin. Analysis of 56 RMs showed that DP cells (i) significantly increase following SIV infection, (ii) are reduced after 12 months of ART in comparison to pre-ART levels, and (iii) expand to a significantly higher frequency following ART interruption. Quantification of total SIV-gag DNA on sorted DP cells from chronically infected RMs showed that these cells are susceptible to SIV infection. These data reinforce earlier observations that CD20+ T cells are infected and expanded by HIV infection, while suggesting that these cells phenotypically overlap activated CD4+ TFH cells that acquire CD20 expression via trogocytosis and can be targeted as part of therapeutic strategies aimed at HIV remission. IMPORTANCE The HIV reservoir is largely composed of latently infected memory CD4+ T cells that persist during antiretroviral therapy and constitute a major barrier toward HIV eradication. In particular, CD4+ T follicular helper cells have been demonstrated as key targets for viral replication and persistence under ART. In lymph nodes from HIV-infected humans and SIV-infected rhesus macaques, we show that CD3+ CD20+ lymphocytes emerge after membrane exchange between T cells and B cells and are enriched in phenotypic, functional, and gene expression profiles found in T follicular helper cells. Furthermore, in SIV-infected rhesus macaques, these cells expand following experimental infection and after interruption of ART and harbor SIV DNA at levels similar to those found in CD4+ T cells; thus, CD3+ CD20+ lymphocytes are susceptible to SIV infection and can contribute to SIV persistence.

Characterization of the Impact of Merkel Cell Polyomavirus-Induced Interferon Signaling on Viral Infection.

Merkel cell polyomavirus (MCPyV) has been associated with approximately 80% of Merkel cell carcinoma (MCC), an aggressive and increasingly incident skin cancer. The link between host innate immunity, viral load control, and carcinogenesis has been established but poorly characterized. We previously established the importance of the STING and NF-κB pathways in the host innate immune response to viral infection. In this study, we further discovered that MCPyV infection of human dermal fibroblasts (HDFs) induces the expression of type I and III interferons (IFNs), which in turn stimulate robust expression of IFN-stimulated genes (ISGs). Blocking type I IFN downstream signaling using an IFN-ß antibody, JAK inhibitors, and CRISPR knockout of the receptor dramatically repressed MCPyV infection-induced ISG expression but did not significantly restore viral replication activities. These findings suggest that IFN-mediated induction of ISGs in response to MCPyV infection is not crucial to viral control. Instead, we found that type I IFN exerts a more direct effect on MCPyV infection postentry by repressing early viral transcription. We further demonstrated that growth factors normally upregulated in wounded or UV-irradiated human skin can significantly stimulate MCPyV gene expression and replication. Together, these data suggest that in healthy individuals, host antiviral responses, such as IFN production induced by viral activity, may restrict viral propagation to reduce MCPyV burden. Meanwhile, growth factors induced by skin abrasion or UV irradiation may stimulate infected dermal fibroblasts to promote MCPyV propagation. A delicate balance of these mutually antagonizing factors provides a mechanism to support persistent MCPyV infection. IMPORTANCE Merkel cell carcinoma is an aggressive skin cancer that is particularly lethal to immunocompromised individuals. Though rare, MCC incidence has increased significantly in recent years. There are no lasting and effective treatments for metastatic disease, highlighting the need for additional treatment and prevention strategies. By investigating how the host innate immune system interfaces with Merkel cell polyomavirus, the etiological agent of most of these cancers, our studies identified key factors necessary for viral control, as well as conditions that support viral propagation. These studies provide new insights for understanding how the virus balances the effects of the host immune defenses and of growth factor stimulation to achieve persistent infection. Since virus-positive MCC requires the expression of viral oncogenes to survive, our observation that type I IFN can repress viral oncogene transcription indicates that these cytokines could be explored as a viable therapeutic option for treating patients with virus-positive MCC.

Low expression of PGC-1ß and other mitochondrial biogenesis modulators in melanoma is associated with growth arrest and the induction of an immunosuppressive gene expression program dependent on MEK and IRF-1.

Mitochondria are specialized metabolic and immune organelles that have important roles in tumor progression, metastasis, and response to chemotherapy and immunotherapy. Mitochondrial biogenesis and functions are under the control of the peroxisome-proliferator activated receptor-gamma (PGC-1) transcriptional coactivators. Recent research unveiled the role of PGC-1α in bolstering mitochondrial oxidative functions and in the suppression of metastasis in melanoma, but the role of PGC-1s in tumor immunology remains elusive. Herein, we show that low PGC-1s expression in human melanoma tumors is associated with increased expression of a repertoire of immunosuppressive (CD73, PD-L2, Galectin-9) and pro-inflammatory (IL-8, TNF, IL-1ß) transcripts, and that experimental depletion of PGC-1ß recapitulates this signature in human melanoma cell lines. The depletion of PGC-1ß reduces the expression of HSPA9, impairs mitochondrial activity, and leads to cell cycle arrest. Using pharmacological and gene silencing approaches, we further show that MEK1/2 and IRF-1 mediate the observed immune transcriptional response. Overall, this research suggests that mitochondrial biogenesis modulators can modulate tumor progression, immune evasion, and response to therapeutics through transcriptional control of immune pathways.

Virus-specific editing identification approach reveals the landscape of A-to-I editing and its impacts on SARS-CoV-2 characteristics and evolution.

Upon SARS-CoV-2 infection, viral intermediates specifically activate the IFN response through MDA5-mediated sensing and accordingly induce ADAR1 p150 expression, which might lead to viral A-to-I RNA editing. Here, we developed an RNA virus-specific editing identification pipeline, surveyed 7622 RNA-seq data from diverse types of samples infected with SARS-CoV-2, and constructed an atlas of A-to-I RNA editing sites in SARS-CoV-2. We found that A-to-I editing was dynamically regulated, varied between tissue and cell types, and was correlated with the intensity of innate immune response. On average, 91 editing events were deposited at viral dsRNA intermediates per sample. Moreover, editing hotspots were observed, including recoding sites in the spike gene that affect viral infectivity and antigenicity. Finally, we provided evidence that RNA editing accelerated SARS-CoV-2 evolution in humans during the epidemic. Our study highlights the ability of SARS-CoV-2 to hijack components of the host antiviral machinery to edit its genome and fuel its evolution, and also provides a framework and resource for studying viral RNA editing.

Massive digital gene expression analysis reveals different predictive profiles for immune checkpoint inhibitor therapy between adenocarcinoma and squamous cell carcinoma of advanced lung cancer.

BACKGROUND: Immune checkpoint inhibitors prolong the survival of non-small cell lung cancer (NSCLC) patients. Although it has been acknowledged that there is some correlation between the efficacy of anti-programmed cell death-1 (PD-1) antibody therapy and immunohistochemical analysis, this technique is not yet considered foolproof for predicting a favorable outcome of PD-1 antibody therapy. We aimed to predict the efficacy of nivolumab based on a comprehensive analysis of RNA expression at the gene level in advanced NSCLC. METHODS: This was a retrospective study on patients with NSCLC who were administered nivolumab at the Kansai Medical University Hospital. To identify genes associated with response to anti-PD-1 antibodies, we grouped patients into responders (complete and partial response) and non-responders (stable and progressive disease) to nivolumab therapy. Significant genes were then identified for these groups using Welch's t-test. RESULTS: Among 42 analyzed cases (20 adenocarcinomas and 22 squamous cell carcinomas), enhanced expression of MAGE-A4, BBC3, and OTOA genes was observed in responders with adenocarcinoma, and enhanced expression of DAB2, HLA-DPB,1 and CDH2 genes was observed in responders with squamous cell carcinoma. CONCLUSIONS: This study predicted the efficacy of nivolumab based on a comprehensive analysis of mRNA expression at the gene level in advanced NSCLC. We also revealed different gene expression patterns as predictors of the effectiveness of anti PD-1 antibody therapy in adenocarcinoma and squamous cell carcinoma.

Variation of TNF modulates cellular immunity of gregarious and solitary locusts against fungal pathogen Metarhizium anisopliae.

Changes in population density lead to phenotypic differentiation of solitary and gregarious locusts, which display different resistance to fungal pathogens; however, how to regulate their cellular immune strategies remains unknown. Here, our stochastic simulation of pathogen proliferation suggested that humoral defense always enhanced resistance to fungal pathogens, while phagocytosis sometimes reduced defense against pathogens. Further experimental data proved that gregarious locusts had significantly decreased phagocytosis of hemocytes compared to solitary locusts. Additionally, transcriptional analysis showed that gregarious locusts promoted immune effector expression (gnbp1 and dfp) and reduced phagocytic gene expression (eater) and the cytokine tumor necrosis factor (TNF). Interestingly, higher expression of the cytokine TNF in solitary locusts simultaneously promoted eater expression and inhibited gnbp1 and dfp expression. Moreover, inhibition of TNF increased the survival of solitary locusts, and injection of TNF decreased the survival of gregarious locusts after fungal infection. Therefore, our results indicate that the alerted expression of TNF regulated the immune strategy of locusts to adapt to environmental changes.

An instructive role for Interleukin-7 receptor α in the development of human B-cell precursor leukemia.

Kinase signaling fuels growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Yet its role in leukemia initiation is unclear and has not been shown in primary human hematopoietic cells. We previously described activating mutations in interleukin-7 receptor alpha (IL7RA) in poor-prognosis "ph-like" BCP-ALL. Here we show that expression of activated mutant IL7RA in human CD34+ hematopoietic stem and progenitor cells induces a preleukemic state in transplanted immunodeficient NOD/LtSz-scid IL2Rγnull mice, characterized by persistence of self-renewing Pro-B cells with non-productive V(D)J gene rearrangements. Preleukemic CD34+CD10highCD19+ cells evolve into BCP-ALL with spontaneously acquired Cyclin Dependent Kinase Inhibitor 2 A (CDKN2A) deletions, as commonly observed in primary human BCP-ALL. CRISPR mediated gene silencing of CDKN2A in primary human CD34+ cells transduced with activated IL7RA results in robust development of BCP-ALLs in-vivo. Thus, we demonstrate that constitutive activation of IL7RA can initiate preleukemia in primary human hematopoietic progenitors and cooperates with CDKN2A silencing in progression into BCP-ALL.

Immunoexpression of PTEN, HER2/neu, and Ki-67 in endoscopic gastric carcinoma biopsies; their correlation with histological subtypes and one-year survival.

BACKGROUND: Gastric carcinoma is a major cause of cancer-related morbidity and mortality worldwide. Gastric neoplasms arise from genetic and epigenetic changes in various genes. Present study evaluates the immunoexpression of PTEN, HER2/neu, and Ki-67 in endoscopic gastric carcinoma biopsies and correlates the expression of these proteins with clinicopathological features. MATERIAL AND METHODS: Adequate endoscopic biopsies of 27 cases of gastric carcinoma were evaluated for World Health Organization (WHO) and Lauren's classification subtypes along with HER2/neu, PTEN, and Ki-67 immunoexpression. HER2/neu immunostaining was scored as proposed in the Trastuzumab for gastric cancer (ToGA) trial while PTEN staining and downregulation were assessed using an immunoreactive score. The cut-off for Ki-67 expression was taken as 90th percentile of the values in adjacent non-neoplastic tissue. All statistical analysis was done at 5% level of significance with SPSS v22 statistical software. RESULTS: Tubular adenocarcinoma was the commonest WHO histological subtype and 56% of cases were of intestinal type as per Lauren's classification. 55.6% of cases showed a complete loss of PTEN expression in neoplastic tissue. 17 of the 19 cases with adjacent non-neoplastic tissue showed PTEN downregulation in neoplastic tissue. 81.5% of cases had a high Ki-67 index and HER2/neu overexpression was noted in 36% of cases. All the four cases who died had high Ki-67 proliferation indices; 3 patients had loss of PTEN expression and HER2/neu overexpression. CONCLUSION: We conclude that these immunomarkers can play important role in the behavior of gastric carcinomas and can be targeted for new therapies.

Complement Factor H-Related 3 Enhanced Inflammation and Complement Activation in Human RPE Cells.

Complement Factor H-Related 3 (FHR-3) is a major regulator of the complement system, which is associated with different diseases, such as age-related macular degeneration (AMD). However, the non-canonical local, cellular functions of FHR-3 remained poorly understood. Here, we report that FHR-3 bound to oxidative stress epitopes and competed with FH for interaction. Furthermore, FHR-3 was internalized by viable RPE cells and modulated time-dependently complement component (C3, FB) and receptor (C3aR, CR3) expression of human RPE cells. Independently of any external blood-derived proteins, complement activation products were detected. Anaphylatoxin C3a was visualized in treated cells and showed a translocation from the cytoplasm to the cell membrane after FHR-3 exposure. Subsequently, FHR-3 induced a RPE cell dependent pro-inflammatory microenvironment. Inflammasome NLRP3 activation and pro-inflammatory cytokine secretion of IL-1ß, IL-18, IL-6 and TNF-α were induced after FHR-3-RPE interaction. Our previously published monoclonal anti-FHR-3 antibody, which was chimerized to reduce immunogenicity, RETC-2-ximab, ameliorated the effect of FHR-3 on ARPE-19 cells. Our studies suggest FHR-3 as an exogenous trigger molecule for the RPE cell "complosome" and as a putative target for a therapeutic approach for associated degenerative diseases.

Tracing TET1 expression in prostate cancer: discovery of malignant cells with a distinct oncogenic signature.

BACKGROUND: Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) is involved in DNA demethylation and transcriptional regulation, plays a key role in the maintenance of stem cell pluripotency, and is dysregulated in malignant cells. The identification of cancer stem cells (CSCs) driving tumor growth and metastasis is the primary objective of biomarker discovery in aggressive prostate cancer (PCa). In this context, we analyzed TET1 expression in PCa. METHODS: A large-scale immunohistochemical analysis of TET1 was performed in normal prostate (NOR) and PCa using conventional slides (50 PCa specimens) and tissue microarrays (669 NOR and 1371 PCa tissue cores from 371 PCa specimens). Western blotting, RT-qPCR, and 450 K methylation array analyses were performed on PCa cell lines. Genome-wide correlation, gene regulatory network, and functional genomics studies were performed using publicly available data sources and bioinformatics tools. RESULTS: In NOR, TET1 was exclusively expressed in normal cytokeratin 903 (CK903)-positive basal cells. In PCa, TET1 was frequently detected in alpha-methylacyl-CoA racemase (AMACR)-positive tumor cell clusters and was detectable at all tumor stages and Gleason scores. Pearson's correlation analyses of PCa revealed 626 TET1-coactivated genes (r > 0.5) primarily encoding chromatin remodeling and mitotic factors. Moreover, signaling pathways regulating antiviral processes (62 zinc finger, ZNF, antiviral proteins) and the pluripotency of stem cells were activated. A significant proportion of detected genes exhibited TET1-correlated promoter hypomethylation. There were 161 genes encoding transcription factors (TFs), of which 133 were ZNF-TFs with promoter binding sites in TET1 and in the vast majority of TET1-coactivated genes. CONCLUSIONS: TET1-expressing cells are an integral part of PCa and may represent CSCs with oncogenic potential.

Nanoconfinement of microvilli alters gene expression and boosts T cell activation.

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanism by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200-nm pores but not in 400-nm pores. Consequently, formation of TCR nanoclustered hotspots within 200-nm pores allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.

UMP-CMP kinase 2 gene expression in macrophages is dependent on the IRF3-IFNAR signaling axis.

Toll-like receptors (TLRs) are highly-conserved pattern recognition receptors that mediate innate immune responses to invading pathogens and endogenous danger signals released from damaged and dying cells. Activation of TLRs trigger downstream signaling cascades, that culminate in the activation of interferon regulatory factors (IRFs), which subsequently leads to type I interferon (IFN) response. In the current study, we sought to expand the scope of gene expression changes in THP1-derived macrophages upon TLR4 activation and to identify interferon-stimulated genes. RNA-seq analysis led to the identification of several known and novel differentially expressed genes, including CMPK2, particularly in association with type I IFN signaling. We performed an in-depth characterization of CMPK2 expression, a nucleoside monophosphate kinase that supplies intracellular UTP/CTP for nucleic acid synthesis in response to type I IFN signaling in macrophages. CMPK2 was significantly induced at both RNA and protein levels upon stimulation with TLR4 ligand-LPS and TLR3 ligand-Poly (I:C). Confocal microscopy and subcellular fractionation indicated CMPK2 localization in both cytoplasm and mitochondria of THP-1 macrophages. Furthermore, neutralizing antibody-based inhibition of IFNAR receptor in THP-1 cells and BMDMs derived from IFNAR KO and IRF3 KO knockout mice further revealed that CMPK2 expression is dependent on LPS/Poly (I:C) mediated IRF3- type I interferon signaling. In summary, our findings suggest that CMPK2 is a potential interferon-stimulated gene in THP-1 macrophages and that CMPK2 may facilitate IRF3- type I IFN-dependent anti-bacterial and anti-viral roles.

HCV Core Protein Induces Chemokine CCL2 and CXCL10 Expression Through NF-κB Signaling Pathway in Macrophages.

HCV core protein is the first structural protein synthesized during hepatitis C virus (HCV) infection and replication. It is released from virus infected liver cells and mediates multiple functions to affect host cell response. The innate immune response is the first line of defense against viral infection. After HCV infection, Kupffer cells (KCs) which are liver macrophages play an important role in host innate immune response. Kupffer cells act as phagocytes and release different cytokines and chemokines to counter viral infection and regulate inflammation and fibrosis in liver. Earlier, we have demonstrated that HCV core protein interacts with gC1qR and activates MAPK, NF-κB and PI3K/AKT pathways in macrophages. In this study, we explored the effect of HCV core protein on CCL2 and CXCL10 expression in macrophages and the signaling pathways involved. Upon silencing of gC1qR, we observed a significant decrease expression of CCL2 and CXCL10 in macrophages in the presence of HCV core protein. Inhibiting NF-κB pathway, but not P38, JNK, ERK and AKT pathways greatly reduced the expression of CCL2 and CXCL10. Therefore, our results indicate that interaction of HCV core protein with gC1qR could induce CCL2 and CXCL10 secretion in macrophages via NF-κB signaling pathway. These findings may shed light on the understanding of how leukocytes migrate into the liver and exaggerate host-derived immune responses and may provide novel therapeutic targets in HCV chronic inflammation.

NF-κB-An Important Player in Xenoestrogen Signaling in Immune Cells.

The proper functioning of the immune system is critical for an effective defense against pathogenic factors such as bacteria and viruses. All the cellular processes taking place in an organism are strictly regulated by an intracellular network of signaling pathways. In the case of immune cells, the NF-κB pathway is considered the key signaling pathway as it regulates the expression of more than 200 genes. The transcription factor NF-κB is sensitive to exogenous factors, such as xenoestrogens (XEs), which are compounds mimicking the action of endogenous estrogens and are widely distributed in the environment. Moreover, XE-induced modulation of signaling pathways may be crucial for the proper development of the immune system. In this review, we summarize the effects of XEs on the NF-κB signaling pathway. Based on our analysis, we constructed a model of XE-induced signaling in immune cells and found that in most cases XEs activate NF-κB. Our analysis indicated that the indirect impact of XEs on NF-κB in immune cells is related to the modulation of estrogen signaling and other pathways such as MAPK and JAK/STAT. We also summarize the role of these aspects of signaling in the development and further functioning of the immune system in this paper.

Expression of the axon guidance factor Slit2 and its receptor Robo1 in patients with Hirschsprung disease: An observational study.

ABSTRACT: Hirschsprung disease (HD) is a common form of digestive tract malformation in children. However, the pathogenesis of HD is not very clear. This study aimed to investigate the expression of slit guidance ligand 2 (Slit2) and roundabout 1 (Robo1) in patients with HD.From January 2018 to January 2019, 30 colon specimens from children with HD undergoing surgical resection at the Department of Surgery in Qilu Children's Hospital of Shandong University were obtained. These specimens were divided into the normal segment group, the transitional segment group and the spastic segment group. Immunohistochemical staining, Western blotting, and real-time polymerase chain reaction were used to measure the expression of Slit2 and Robo1 in the intestinal walls of normal, transitional, and spastic segments.Immunohistochemical staining and Western blot analyses showed high levels of the Slit2 and Robo1 proteins in normal ganglion cells in children with HD, lower levels in transitional ganglion cells, and no expression in spastic segments, with significant differences between groups (P < .05). Similarly, the real-time polymerase chain reaction results were consistent with the Western blot analysis results.The expression of Slit2 and Robo1 decreases significantly in the spastic segment of the intestinal tract in patients with HD.

Differential responses of chicken monocyte-derived dendritic cells infected with Salmonella Gallinarum and Salmonella Typhimurium.

Salmonella enterica serovar Gallinarum is a host-restricted bacterial pathogen that causes a serious systemic disease exclusively in birds of all ages. Salmonella enterica serovar Typhimurium is a host-generalist serovar. Dendritic cells (DCs) are key antigen-presenting cells that play an important part in Salmonella host-restriction. We evaluated the differential response of chicken blood monocyte-derived dendritic cells (chMoDCs) exposed to S. Gallinarum or S. Typhimurium. S. Typhimurium was found to be more invasive while S. Gallinarum was more cytotoxic at the early phase of infection and later showed higher resistance against chMoDCs killing. S. Typhimurium promoted relatively higher upregulation of costimulatory and other immune function genes on chMoDCs in comparison to S. Gallinarum during early phase of infection (6 h) as analyzed by real-time PCR. Both Salmonella serovars strongly upregulated the proinflammatory transcripts, however, quantum was relatively narrower with S. Gallinarum. S. Typhimurium-infected chMoDCs promoted relatively higher proliferation of naïve T-cells in comparison to S. Gallinarum as assessed by mixed lymphocyte reaction. Our findings indicated that host restriction of S. Gallinarum to chicken is linked with its profound ability to interfere the DCs function. Present findings provide a valuable roadmap for future work aimed at improved vaccine strategies against this pathogen.

Early p38 Activation Regulated by MKP-1 Is Determinant for High Levels of IL-10 Expression Through TLR2 Activation.

Toll-like receptors (TLRs) play a crucial role in the recognition of pathogen-derived components as a first line of defense against infections. It has been suggested that depending on the nature of the pathogens, TLRs activation induce a distinct cytokine profile that may contribute to the polarization of the acquired immune response. Here, we investigated the early MAPK signaling activation via TLR4 and TLR2 receptors and its impact in differential cytokine profile by macrophages. We found that TLR2 ligands activated MAPKs p38 and ERK earlier compared to the TLR4 ligand LPS in macrophages. Higher IL-10/IL-12 and IL-10/TNF-α ratios were also observed at later time points in response to TLR2 ligands compared to LPS. The results also indicate an earlier activation of the phosphatase MKP-1 and that MKP-1 KO macrophages show a prolongation in p38 phosphorylation in response to TLR2 stimulation. Furthermore, p38 is critical for IL-10 expression in response to TLR2 ligands, which triggers the macrophage change to a M2 and regulatory phenotype in contrast to the M1 phenotype induced by TLR4 activation. Therefore, the early TLR2-mediated p38 induction contributes for the high IL-10 production, likely as a virulence strategy to suppress host Th1 response against certain types of pathogens.

Revealing immune responses in the Mycobacterium avium subsp. paratuberculosis-infected THP-1 cells using single cell RNA-sequencing.

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne's disease, which is a chronic and debilitating disease in ruminants. MAP is also considered to be a possible cause of Crohn's disease in humans. However, few studies have focused on the interactions between MAP and human macrophages to elucidate the pathogenesis of Crohn's disease. We sought to determine the initial responses of human THP-1 cells against MAP infection using single-cell RNA-seq analysis. Clustering analysis showed that THP-1 cells were divided into seven different clusters in response to phorbol-12-myristate-13-acetate (PMA) treatment. The characteristics of each cluster were investigated by identifying cluster-specific marker genes. From the results, we found that classically differentiated cells express CD14, CD36, and TLR2, and that this cell type showed the most active responses against MAP infection. The responses included the expression of proinflammatory cytokines and chemokines such as CCL4, CCL3, IL1B, IL8, and CCL20. In addition, the Mreg cell type, a novel cell type differentiated from THP-1 cells, was discovered. Thus, it is suggested that different cell types arise even when the same cell line is treated under the same conditions. Overall, analyzing gene expression patterns via scRNA-seq classification allows a more detailed observation of the response to infection by each cell type.

Cross-tissue single-cell landscape of human monocytes and macrophages in health and disease.

Mononuclear phagocytes (MNPs) encompass dendritic cells, monocytes, and macrophages (MoMac), which exhibit antimicrobial, homeostatic, and immunoregulatory functions. We integrated 178,651 MNPs from 13 tissues across 41 datasets to generate a MNP single-cell RNA compendium (MNP-VERSE), a publicly available tool to map MNPs and define conserved gene signatures of MNP populations. Next, we generated a MoMac-focused compendium that revealed an array of specialized cell subsets widely distributed across multiple tissues. Specific pathological forms were expanded in cancer and inflammation. All neoplastic tissues contained conserved tumor-associated macrophage populations. In particular, we focused on IL4I1+CD274(PD-L1)+IDO1+ macrophages, which accumulated in the tumor periphery in a T cell-dependent manner via interferon-γ (IFN-γ) and CD40/CD40L-induced maturation from IFN-primed monocytes. IL4I1_Macs exhibited immunosuppressive characteristics through tryptophan degradation and promoted the entry of regulatory T cell into tumors. This integrated analysis provides a robust online-available platform for uniform annotation and dissection of specific macrophage functions in healthy and pathological states.