Immune Stimuli Research Articles

Nanoplatforms Potentiated Ablation-Immune Synergistic Therapy through Improving Local Control and Suppressing Recurrent Metastasis.

Minimally invasive ablation has been widely applied for treatment of various solid tumors, including hepatocellular carcinoma, renal cell carcinoma, breast carcinomas, etc. In addition to removing the primary tumor lesion, ablative techniques are also capable of improving the anti-tumor immune response by inducing immunogenic tumor cell death and modulating the tumor immune microenvironment, which may be of great benefit to inhibit the recurrent metastasis of residual tumor. However, the short-acting activated anti-tumor immunity of post-ablation will rapidly reverse into an immunosuppressive state, and the recurrent metastasis owing to incomplete ablation is closely associated with a dismal prognosis for the patients. In recent years, numerous nanoplatforms have been developed to improve the local ablative effect through enhancing the targeting delivery and combining it with chemotherapy. Particularly, amplifying the anti-tumor immune stimulus signal, modulating the immunosuppressive microenvironment, and improving the anti-tumor immune response with the versatile nanoplatforms have heralded great application prospects for improving the local control and preventing tumor recurrence and distant metastasis. This review discusses recent advances in nanoplatform-potentiated ablation-immune synergistic tumor therapy, focusing on common ablation techniques including radiofrequency, microwave, laser, and high-intensity focused ultrasound ablation, cryoablation, and magnetic hyperthermia ablation, etc. We discuss the advantages and challenges of the corresponding therapies and propose possible directions for future research, which is expected to provide references for improving the traditional ablation efficacy.

Microglia secrete distinct sets of neurotoxins in a stimulus-dependent manner.

Microglia are the resident immune cells of the brain which regulate both the innate and adaptive neuroimmune responses in health and disease. In response to specific endogenous and exogenous stimuli, microglia transition to one of their reactive states characterized by altered morphology and function, including their secretory profile. A component of the microglial secretome is cytotoxic molecules capable of causing damage and death to nearby host cells, thus contributing to the pathogenesis of neurodegenerative disorders. Indirect evidence from secretome studies and measurements of mRNA expression using diverse microglial cell types suggest different stimuli may induce microglia to secrete distinct subsets of cytotoxins. We demonstrate the accuracy of this hypothesis directly by challenging murine BV-2 microglia-like cells with eight different immune stimuli and assessing secretion of four potentially cytotoxic molecules, including nitric oxide (NO), tumor necrosis factor α (TNF), C-X-C motif chemokine ligand 10 (CXCL10), and glutamate. Lipopolysaccharide (LPS) and a combination of interferon (IFN)-γ plus LPS induced secretion of all toxins studied. IFN-β, IFN-γ, polyinosinic:polycytidylic acid (poly I:C), and zymosan A upregulated secretion of subsets of these four cytotoxins. LPS and IFN-γ, alone or in combination, as well as IFN-β induced toxicity of BV-2 cells towards murine NSC-34 neuronal cells, while ATP, N-formylmethionine-leucyl-phenylalanine (fMLP), and phorbol 12-myristate 13-acetate (PMA) did not affect any parameters studied. Our observations contribute to a growing body of knowledge on the regulation of the microglial secretome, which may inform future development of novel therapeutics for neurodegenerative diseases, where dysregulated microglia are key contributors to pathogenesis.

Innate Immune Training Enhances the Reactivation of Latently Infected HIV-1 from Monocytic Cell lines

Abstract HIV virus can persist in a latent but activatable chronic state in resting CD4 +T-cells and macrophages that are more persistent, leading to the requirement of lifelong combination antiretroviral therapy (cART). The latency of HIV-1 in macrophage cells is associated with increased chromatin condensation induced by histone methylation at HIV-1 integration sites. Innate immune training of macrophages has been shown to relieve chromosome condensation through epigenetic rewiring. We hypothesized that training of macrophages could enhance the reactivation of HIV-1 by opening the chromatin to facilitate transcription in response to Latency Reversing Agents (LRAs). We used beta-glucan, Syk inhibitor and Rutaecarpine to train the THP89GFP cell line, which is an experimental model for latently infected HIV-1 monocytes. We found that these trained THP89GFP cells have higher transcription of HIV-1 specific genes in response to PMA stimulation. To check the epigenetic changes associated with training in the proviral HIV-1 DNA of THP89GFP, we performed a chromatin immunoprecipitation for the histone mark, H3K27Ac. Consistent with the higher induction of transcription of HIV-1 genes, we observed that multiple regions of the HIV-1 LTR had higher deposition of H3K27Ac in the trained macrophages. Our studies thus show that induction of trained innate immunity may be a viable approach to the reactivation of latently infected HIV-1. This finding supports the hypothesis that innate immune training stimuli could be developed as novel candidates for enhancing reactivation of latently infected HIV-1, which may facilitate elimination of macrophage reservoirs. This work was supported by the Intramural Research Program of NIAID, NIH This work was supported by the Intramural Research Program of NIAID, NIH and Office of AIDS research.

Human platelets release amyloid peptides β1-40 and β1-42 in response to hemostatic, immune, and hypoxic stimuli

BackgroundPlatelets contain high levels of amyloid β (Aβ) peptides and have been suggested to participate in the deposition of amyloid plaques in Alzheimer’s disease. ObjectivesThis study aimed to determine whether human platelets release pathogenic Aβ peptides Aβ1-42 and Aβ1-40 and to characterize the mechanisms regulating this phenomenon. MethodsEnzyme-linked immunosorbent assays (ELISAs) and single platelet immunostaining were utilised to study the release and subcellular localisation of Aβ, respectively. ResultsELISAs revealed that thrombin and the pro-inflammatory molecule lipopolysaccharide (LPS) induce platelet release of both Aβ1-42 and Aβ1-40. Notably, LPS preferentially induced the release of Aβ1-42, which was potentiated by the reduction of oxygen from atmospheric levels to physiological hypoxia. The selective β secretase (BACE) inhibitor LY2886721 showed no effect on the release of either Aβ1-40 or Aβ1-42 in our ELISA experiments. This suggested a store-and-release mechanism that was confirmed in immunostaining experiments showing co-localisation of cleaved Aβ peptides with platelet alpha granules. ConclusionTaken together, our data suggest that human platelets release pathogenic Aβ peptides as a result of a store-and-release mechanism rather than a de novo proteolytic event. Although further studies are required to fully characterize this phenomenon, we suggest the possibility of a role for platelets in the deposition of Aβ peptides and the formation of amyloid plaques. Interestingly, the combination of hypoxia and inflammation that we simulated in vitro with reduced oxygen tension and LPS may increase the release of fibrillogenic Aβ1-42 and, consequently, exacerbate amyloid plaque deposition in the brain of patients with Alzheimer’s disease.

The human P2X7 receptor alters microglial morphology and cytokine secretion following immunomodulation.

Introduction: In recent years, purinergic signaling via the P2X7 receptor (P2X7R) on microglia has repeatedly been implicated in depression genesis. However, it remains unclear which role the human P2X7R (hP2X7R) plays in regulating both microglia morphology and cytokine secretion upon different environmental and immune stimuli, respectively. Methods: For this purpose, we used primary microglial cultures derived from a humanized microglia-specific conditional P2X7R knockout mouse line to emulate different gene-environment interactions between microglial hP2X7R and molecular proxies of psychosocial and pathogen-derived immune stimuli. Microglial cultures were subjected to treatments with the agonists 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP) and lipopolysaccharides (LPS) combined with specific P2X7R antagonists (JNJ-47965567, A-804598). Results: Morphotyping revealed overall high baseline activation due to the in vitro conditions. Both BzATP and LPS + BzATP treatment increased round/ameboid microglia and decreased polarized and ramified morphotypes. This effect was stronger in hP2X7R-proficient (CTRL) compared to knockout (KO) microglia. Aptly, we found antagonism with JNJ-4796556 and A-804598 to reduce round/ameboid microglia and increase complex morphologies only in CTRL but not KO microglia. Single cell shape descriptor analysis confirmed the morphotyping results. Compared to KO microglia, hP2X7R-targeted stimulation in CTRLs led to a more pronounced increase in microglial roundness and circularity along with an overall higher decrease in aspect ratio and shape complexity. JNJ-4796556 and A-804598, on the other hand, led to opposite dynamics. In KO microglia, similar trends were observed, yet the magnitude of responses was much smaller. Parallel assessment of 10 cytokines demonstrated the proinflammatory properties of hP2X7R. Following LPS + BzATP stimulation, IL-1β, IL-6, and TNFα levels were found to be higher and IL-4 levels lower in CTRL than in KO cultures. Vice versa, hP2X7R antagonists reduced proinflammatory cytokine levels and increased IL-4 secretion. Discussion: Taken together, our results help disentangle the complex function of microglial hP2X7R downstream of various immune stimuli. In addition, this is the first study in a humanized, microglia-specific in vitro model identifying a so far unknown potential link between microglial hP2X7R function and IL-27 levels.

5613426 LOW CHRONIC INFLAMMATION IN SCD

Introduction: Sickle Cell Disease (SCD) is a genetic disorder of hemoglobin affecting 30 million people worldwide in which the mutant HbS polymerize upon deoxygenation, causing the eritrocyte to assume a sickle morphology and reduce the fluidity of cell membrane, adhering to leucocyte immobilized to the endothelium, causing microvascular occlusion, vaso occlusive crisis and tissue ischemia (1). In additon to eritrocyte sickling, one of the hallmarks of SCD is the continuous presence of basal inflammatory process. SC patients report the following condition: higher baseline leucocyte count (2) Higher level of soluble CD62L (3) neutrofils express higher activation molecules, CD64 (4), CD11b/CD18 (5) monocytes demonstrate activated phenotypes and higer propensity to IL-1beta and TNF-alfa (6) platelets are chronically activated and express higher level of CD40L (7) patience exhibit increased intestinal permeability related to enterocyte injury (8) gut disbiosis that is associated with hospitalization and VOC (9) Ongoing inflammatory process not only render SCD patience to developing VOC, they also create vicious cicle of the pathology that represent a significant risk in the progression of the disease and in chronic damage tissue. Aims: While advance have been made to reduce the frequency of VOC in SCD, there have been very little change in the management of patience during symptom-free “steady state”. The results from various studies on treating VOC using monotherapy have so far been universally disappointing. This is not surprising in view of the complex nature of VOC. Successful treatment of VOC targeting of the downstream immunological pathways requires multipronged approaches using combination therapy, just like treatment of malignant diseases using combination chemotherapy. Methods: The reviwes of the scientific litterature is full of interesting articles in which is enlighted the interaction between the immune system and different stimuli in order to obtain a modulation of the immune reasponse toward a tolerogenic way. Results: In PubMed there are very interesting report that investigate the approach to the immune system in SCD in order to reduce the basal inflammation. There are many way to interact with the immune system to promote a tolerogenic way in SCD such as: supplement of Vitamin D to reduce resporatory illness (10) supplement of Omega-3 to reduce CRP (11) supplement of Glutamine to reduce hospitalization and pain crises (12) probiotic or enterosorbent can reduce gut disbiosis and ameliorate clinical outcome (13) moderate exercise improve cardiopulmonary function and quality life (14) (15) Conclusion: SCD is a genetic disorder caracterized by a continuous presence of low chronic inflammation and studies in human have found that in SCD patience, the more the immune system is activated, the more are likely to get involved in clinical complication such alloimmunization, hospitalization and pain crises (16). Recent evidence suggest that bioregulatory approach toward a tolerogenic way of the immune response could lead to an amelioration of clinical outcome and improvement of quality of life with cheap and feasible approaches.

Inhibition of phosphoinositide‑3 kinases γ/δ ameliorates pulmonary granuloma by rescuing Treg function in a sarcoidosis model.

Sarcoidosis is a multisystem inflammatory disease characterized by the development of Th1/Th17/regulatory T cells (Tregs)-related non-caseating granulomas. Phosphoinositide-3 kinases δ/γ (PI3Kδ/γ) play an important role in the maintenance of effective immunity, especially for Tregs homeostasis and stability. In the present study, superoxide dismutase A (SodA) stimulation was used to establish the sarcoidosis mouse model. The second immune stimulus was accompanied by CAL-101 (PI3Kδ inhibitor) or AS-605240 (PI3Kδ/γ inhibitor) treatment. To detect the effect of the PI3Kδ/γ inhibitor on the morphology of pulmonary granuloma and the activation of the PI3K signaling pathway, hematoxylin and eosin staining and immunofluorescence and western blotting was used, respectively. Fluorescence-activated cell sorting analysis and reverse transcription-quantitative PCR were adopted to detect the effect of the PI3Kδ/γ inhibitor on the SodA-induced sarcoidosis mouse model in respect to immune cell disorder and the function of Treg cells, with CD4+CD25- T cells and CD4+CD25+ T cells sorted by magnetic cell sorting. The results demonstrated that the inhibition of PI3Kδ/γ by transtracheal CAL-101/AS-605240 administration facilitated pulmonary granuloma formation. These therapeutic effects were associated with certain mechanisms, including suppressing the aberrantly activated PI3K/Akt signaling in both pulmonary granuloma and Tregs, particularly rescuing the suppressive function of Tregs. Notably, CAL-101 was more effective in immune modulation compared with AS-605240 and could overcome the aberrantly activated Akt in the lung and Tregs. These results suggest that PI3K/Akt signaling, especially the PI3Kδ subunit, can play a key role in optimal Tregs-mediated protection against pulmonary sarcoidosis. Therefore, transtracheal usage of PI3Kδ/γ inhibitors is an attractive therapy that may be developed into a new immune-therapeutic principle for sarcoidosis in the future.

Host-derived mannose glycans trigger a pathogenic γδ T cell/IL-17a axis in autoimmunity.

Autoimmune diseases are life-threatening disorders that cause increasing disability over time. Systemic lupus erythematosus (SLE) and other autoimmune diseases arise when immune stimuli override mechanisms of self-tolerance. Accumulating evidence has demonstrated that protein glycosylation is substantially altered in autoimmune disease development, but the mechanisms by which glycans trigger these autoreactive immune responses are still largely unclear. In this study, we found that presence of microbial-associated mannose structures at the surface of the kidney triggers the recognition of DC-SIGN-expressing γδ T cells, inducing a pathogenic interleukin-17a (IL-17a)-mediated autoimmune response. Mice lacking Mgat5, which have a higher abundance of mannose structures in the kidney, displayed increased γδ T cell infiltration into the kidney that was associated with spontaneous development of lupus in older mice. N-acetylglucosamine supplementation, which promoted biosynthesis of tolerogenic branched N-glycans in the kidney, was found to inhibit γδ T cell infiltration and control disease development. Together, this work reveals a mannose-γδ T cell-IL-17a axis in SLE immunopathogenesis and highlights glycometabolic reprogramming as a therapeutic strategy for autoimmune disease treatment.

The Role of B Cells in Skin Inflammation

The role of B cells in skin inflammation highlights the versatility of B cells in their response to diverse immune stimuli perceived as external threats to the host. B cells play a vital role in the immune system’s ability to effectively respond to offenses to the healthy skin microenvironment, such as wounds, malignant tumors, vector (tick)-borne pathogens, and the development of red meat allergies after a tick bite. Select examples from the literature that illustrate the role of B cells in these disrupted states reviewed individually below, and highlight little overlap in the role B cells play in the different diseases. Future studies will need to continue to research the relatively newfound role of B cells in maintaining healthy skin.

Insights into dendritic cell maturation during infection with application of advanced imaging techniques.

Dendritic cells (DCs) are crucial for the initiation and regulation of adaptive immune responses. When encountering immune stimulus such as bacterial and viral infection, parasite invasion and dead cell debris, DCs capture antigens, mature, acquire immunostimulatory activity and transmit the immune information to naïve T cells. Then activated cytotoxic CD8+ T cells directly kill the infected cells, while CD4+ T helper cells release cytokines to aid the activity of other immune cells, and help B cells produce antibodies. Thus, detailed insights into the DC maturation process are necessary for us to understand the working principle of immune system, and develop new medical treatments for infection, cancer and autoimmune disease. This review summarizes the DC maturation process, including environment sensing and antigen sampling by resting DCs, antigen processing and presentation on the cell surface, DC migration, DC-T cell interaction and T cell activation. Application of advanced imaging modalities allows visualization of subcellular and molecular processes in a super-high resolution. The spatiotemporal tracking of DCs position and migration reveals dynamics of DC behavior during infection, shedding novel lights on DC biology.

Invited Perspective: Environmental Chemical-Sensing AHR Remains an Enigmatic Key Player in Toxicology.

Invited Perspective: Environmental Chemical-Sensing AHR Remains an Enigmatic Key Player in Toxicology.

Systems Biology Approaches for the Improvement of Oncolytic Virus-Based Immunotherapies.

Oncolytic virus (OV)-based immunotherapy is mainly dependent on establishing an efficient cell-mediated antitumor immunity. OV-mediated antitumor immunity elicits a renewed antitumor reactivity, stimulating a T-cell response against tumor-associated antigens (TAAs) and recruiting natural killer cells within the tumor microenvironment (TME). Despite the fact that OVs are unspecific cancer vaccine platforms, to further enhance antitumor immunity, it is crucial to identify the potentially immunogenic T-cell restricted TAAs, the main key orchestrators in evoking a specific and durable cytotoxic T-cell response. Today, innovative approaches derived from systems biology are exploited to improve target discovery in several types of cancer and to identify the MHC-I and II restricted peptide repertoire recognized by T-cells. Using specific computation pipelines, it is possible to select the best tumor peptide candidates that can be efficiently vectorized and delivered by numerous OV-based platforms, in order to reinforce anticancer immune responses. Beyond the identification of TAAs, system biology can also support the engineering of OVs with improved oncotropism to reduce toxicity and maintain a sufficient portion of the wild-type virus virulence. Finally, these technologies can also pave the way towards a more rational design of armed OVs where a transgene of interest can be delivered to TME to develop an intratumoral gene therapy to enhance specific immune stimuli.

β-D-XYLOSIDASE 4 modulates systemic immune signaling in Arabidopsis thaliana.

Pectin- and hemicellulose-associated structures of plant cell walls participate in defense responses against pathogens of different parasitic lifestyles. The resulting immune responses incorporate phytohormone signaling components associated with salicylic acid (SA) and jasmonic acid (JA). SA plays a pivotal role in systemic acquired resistance (SAR), a form of induced resistance that - after a local immune stimulus - confers long-lasting, systemic protection against a broad range of biotrophic invaders. β-D-XYLOSIDASE 4 (BXL4) protein accumulation is enhanced in the apoplast of plants undergoing SAR. Here, two independent Arabidopsis thaliana mutants of BXL4 displayed compromised systemic defenses, while local resistance responses to Pseudomonas syringae remained largely intact. Because both phloem-mediated and airborne systemic signaling were abrogated in the mutants, the data suggest that BXL4 is a central component in SAR signaling mechanisms. Exogenous xylose, a possible product of BXL4 enzymatic activity in plant cell walls, enhanced systemic defenses. However, GC-MS analysis of SAR-activated plants revealed BXL4-associated changes in the accumulation of certain amino acids and soluble sugars, but not xylose. In contrast, the data suggest a possible role of pectin-associated fucose as well as of the polyamine putrescine as regulatory components of SAR. This is the first evidence of a central role of cell wall metabolic changes in systemic immunity. Additionally, the data reveal a so far unrecognized complexity in the regulation of SAR, which might allow the design of (crop) plant protection measures including SAR-associated cell wall components.

Alteration of immunoregulatory genes expression in mesenchymal stromal cells upon priming with B18R as an interferon binding protein.

The B18R protein encoded by the Vaccinia virus decoys Type 1 interferons and inhibits the activity of several type I IFN members. In vitro transcription protocols benefit from this molecule's involvement in enhancing cell viability by inhibiting interferon signal transduction. As a result of their immunomodulatory properties and potential to regenerate, mesenchymal stromal cells (MSCs) are increasingly considered an alternative treatment for a wide range of immune disorders. In this study, we investigated the modification of expression of several genes involved in immune-related pathways after preconditioning MSCs with two immune stimuli, including poly(I:C) and LPS. ASCs were isolated and primed with B18R, and after exposure to poly(I:C) and LPS, the expression of the same sets of genes as in the previous experiment was evaluated. Following total RNA isolation from primed cells and cDNA preparation, real-time quantitative PCR was performed for several immunomodulatory and immune-related genes, including IDO1, TDO2, COX-2, TGF- β 1, TNF- α, IL-1 β , IL-6, TLR3, TLR4, and MCP-1. Pretreatment of MSCs with poly(I:C) and LPS significantly increased the expression of all mentioned genes, while upon the B18R challenge followed by poly(I:C) and LPS treatment, they were down-regulated. Finally, it was observed that the relative expression level of IFN -β has significantly decreased in MSCs+B18R+poly(I:C) and LPS in comparison with these groups without B18R. The data indicated that the presence of B18R prevents the overexpression of several immune-related genes, which are overexpressed in the in vitro inflammatory environment.

Chemical mimetics of the N-degron pathway alleviate systemic inflammation by activating mitophagy and immunometabolic remodeling

The Arg/N-degron pathway, which is involved in the degradation of proteins bearing an N-terminal signal peptide, is connected to p62/SQSTM1-mediated autophagy. However, the impact of the molecular link between the N-degron and autophagy pathways is largely unknown in the context of systemic inflammation. Here, we show that chemical mimetics of the N-degron Nt-Arg pathway (p62 ligands) decreased mortality in sepsis and inhibited pathological inflammation by activating mitophagy and immunometabolic remodeling. The p62 ligands alleviated systemic inflammation in a mouse model of lipopolysaccharide (LPS)-induced septic shock and in the cecal ligation and puncture model of sepsis. In macrophages, the p62 ligand attenuated the production of proinflammatory cytokines and chemokines in response to various innate immune stimuli. Mechanistically, the p62 ligand augmented LPS-induced mitophagy and inhibited the production of mitochondrial reactive oxygen species in macrophages. The p62 ligand-mediated anti-inflammatory, antioxidative, and mitophagy-activating effects depended on p62. In parallel, the p62 ligand significantly downregulated the LPS-induced upregulation of aerobic glycolysis and lactate production. Together, our findings demonstrate that p62 ligands play a critical role in the regulation of inflammatory responses by orchestrating mitophagy and immunometabolic remodeling.

Effects of sex and pro-inflammatory cytokines on context discrimination memory

In learning and memory tasks, immune overactivation is associated with impaired performance, while normal immune activation is associated with optimal performance. In one specific domain of memory, context discrimination memory, peripheral immune stimulation has been shown to impair performance on the context-object discrimination memory task in male rats. In order to evaluate potential sex differences in this task, as well as potential mechanisms for the memory impairment, we evaluated the ability of peripheral immune stimulation to impair task performance in both males and females. Next, we examined whether treatment with interleukin-1 receptor antagonist (IL-1ra), a receptor antagonist for the pro-inflammatory cytokine interleukin (IL)−1β, was able to rescue the memory deficit. We examined microglial morphology in the hippocampus and cytokine mRNA and protein expression in the hippocampus and the periphery. Male rats displayed memory impairment in response to LPS, and this impairment was not rescued by IL-1ra. Female rats did not have significant memory impairments and IL-1ra administration improved memory following inflammation. A subset of cytokines and chemokines were increased only in LPS-treated males. Inflammation alone did not alter microglia morphology, but IL-1ra did in certain sub-regions of the hippocampus. Together, these results indicate that sex differences exist in the ability of a peripheral immune stimulus to influence context discrimination memory and specific cytokine signals may be altered in impaired males. This study highlights the importance of sex differences in response to inflammatory challenges, especially related to memory impairments in context discrimination memory.

A nanoadjuvant that dynamically coordinates innate immune stimuli activation enhances cancer immunotherapy and reduces immune cell exhaustion.

Although conventional innate immune stimuli contribute to immune activation, they induce exhausted immune cells, resulting in suboptimal cancer immunotherapy. Here we suggest a kinetically activating nanoadjuvant (K-nanoadjuvant) that can dynamically integrate two waves of innate immune stimuli, resulting in effective antitumour immunity without immune cell exhaustion. The combinatorial code of K-nanoadjuvant is optimized in terms of the order, duration and time window between spatiotemporally activating Toll-like receptor 7/8 agonist and other Toll-like receptor agonists. K-nanoadjuvant induces effector/non-exhausted dendritic cells that programme the magnitude and persistence of interleukin-12 secretion, generate effector/non-exhausted CD8+ T cells, and activate natural killer cells. Treatment with K-nanoadjuvant as a monotherapy or in combination therapy with anti-PD-L1 or liposomes (doxorubicin) results in strong antitumour immunity in murine models, with minimal systemic toxicity, providing a strategy for synchronous and dynamic tailoring of innate immunity for enhanced cancer immunotherapy.

Loss of the Immunomodulatory Transcription Factor BATF2 in Humans Is Associated with a Neurological Phenotype.

Epilepsy and mental retardation are known to be associated with pathogenic mutations in a broad range of genes that are expressed in the brain and have a role in neurodevelopment. Here, we report on a family with three affected individuals whose clinical symptoms closely resemble a neurodevelopmental disorder. Whole-exome sequencing identified a homozygous stop-gain mutation, p.Gln19*, in the BATF2 gene in the patients. The BATF2 transcription factor is predominantly expressed in macrophages and monocytes and has been reported to modulate AP-1 transcription factor-mediated pro-inflammatory responses. Transcriptome analysis showed altered base-level expression of interferon-stimulated genes in the patients' blood, typical for type I interferonopathies. Peripheral blood mononuclear cells from all three patients demonstrated elevated responses to innate immune stimuli, which could be reproduced in CRISPR-Cas9-generated BATF2-/- human monocytic cell lines. BATF2 is, therefore, a novel disease-associated gene candidate for severe epilepsy and mental retardation related to dysregulation of immune responses, which underscores the relevance of neuroinflammation for epilepsy.

Use of CRISPR/CAS9 Technologies to Study the Role of TLR in Dendritic Cell Subsets.

Dendritic cells (DCs) have a significant role in coordinating both innate and adaptive immunity by serving as sentinels that detect invaders and initiate immune responses to eliminate them, as well as presenting antigens to activate adaptive immune responses that are specific to the antigen and the context in which it was detected. The regulation of DC functions is complex and involves intracellular drivers such as transcription factors and signaling pathways, as well as intercellular interactions with adhesion molecules, chemokines, and their receptors in the microenvironment. Toll-like receptors (TLRs) are crucial for DCs to detect pathogen-associated molecular patterns (PAMPs) and initiate downstream signaling pathways that lead to DC maturation and education in bridging with adaptive immunity, including the upregulation of MHC class II expression, induction of CD80, CD86, and CD40, and production of innate cytokines. Understanding the TLR pathways that DCs use to respond to innate immune stimuli and convert them into adaptive responses is important for new therapeutic targets identification.We present a novel platform that offers a fast and affordable CRISPR-Cas9 screening of genes that are involved in dendritic cells' TLR-dependent activation. Using CRISPR/Cas9 screening to target individual TLR genes in different dendritic cell subsets allows the identification of TLR-dependent pathways that regulate dendritic cell activation and cytokine production. This approach offers the efficient targeting of TLR driver genes to modulate the immune response and identify novel immune response regulators, establishing a causal link between these regulators and functional phenotypes based on genotypes.

Open multi-organ communication device for easy interrogation of tissue slices.

Here, we have developed an open multi-organ communication device that facilitates cellular and molecular communication between ex vivo organ slices. Measuring communication between organs is vital for understanding the mechanisms of health regulation yet remains difficult with current technology. Communication between organs along the gut-brain-immune axis is a key regulator of gut homeostasis. As a novel application of the device, we have used tissue slices from the Peyer's patch (PP) and mesenteric lymph node (MLN) due to their importance in gut immunity; however, any organ slices could be used here. The device was designed and fabricated using a combination of 3D printed molds for polydimethylsiloxane (PDMS) soft lithography, PDMS membranes, and track-etch porous membranes. To validate cellular and protein transfer between organs on-chip, we used fluorescence microscopy to quantitate movement of fluorescent proteins and cells from the PP to the MLN, replicating the initial response to immune stimuli in the gut. IFN-γ secretion during perfusion from a naïve vs. inflamed PP to a healthy MLN was quantitated to demonstrate soluble signaling molecules are moving on-chip. Finally, transient catecholamine release was measured during perfusion from PP to MLN using fast-scan cyclic voltammetry at carbon-fiber microelectrodes to demonstrate a novel application of the device for real-time sensing during communication. Overall, we show an open-well multi-organ device capable of facilitating transfer of soluble factors and cells with the added benefit of being available for external analysis techniques like electrochemical sensing which will advance abilities to probe communication in real-time across multiple organs ex vivo.