Enhancing Therapeutic Efficacy of FLT3 Inhibitors with Combination Therapy for Treatment of Acute Myeloid Leukemia.

FMS-like tyrosine kinase 3 (FLT3) mutations are genetic changes found in approximately thirty percent of patients with acute myeloid leukemia (AML). FLT3 mutations in AML represent a challenging clinical scenario characterized by a high rate of relapse, even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The advent of FLT3 tyrosine kinase inhibitors (TKIs), such as midostaurin and gilteritinib, has shown promise in achieving complete remission. However, a substantial proportion of patients still experience relapse following TKI treatment, necessitating innovative therapeutic strategies. This review critically addresses the current landscape of TKI treatments for FLT3+ AML, with a particular focus on gilteritinib. Gilteritinib, a highly selective FLT3 inhibitor, has demonstrated efficacy in targeting the mutant FLT3 receptor, thereby inhibiting aberrant signaling pathways that drive leukemic proliferation. However, monotherapy with TKIs may not be sufficient to eradicate AML blasts. Specifically, we provide evidence for integrating gilteritinib with mammalian targets of rapamycin (mTOR) inhibitors and interleukin-15 (IL-15) complexes. The combination of gilteritinib, mTOR inhibitors, and IL-15 complexes presents a compelling strategy to enhance the eradication of AML blasts and enhance NK cell killing, offering a potential for improved patient outcomes.

HIV-1 infection promotes neuroinflammation and neuron pathogenesis in novel microglia-containing cerebral organoids.

Cerebral organoids (COs) are a valuable tool to study the intricate interplay between glial cells and neurons in brain development and disease, including HIV-associated neuroinflammation. We developed a novel approach to generate microglia containing COs (CO-iMs) by co-culturing hematopoietic progenitors and induced pluripotent stem cells. This approach allowed for the differentiation of microglia within the organoids concomitantly to the neuronal progenitors. CO- iMs exhibited higher efficiency in generation of CD45 + /CD11b + /Iba-1 + microglia cells compared to conventional COs with physiologically relevant proportion of microglia (∼7%). CO-iMs exhibited substantially higher expression of microglial homeostatic and sensome markers as well as markers for the complement cascade. CO-iMs showed susceptibility to HIV infection resulting in a significant increase in several pro-inflammatory cytokines/chemokines and compromised neuronal function, which were abrogated by addition of antiretrovirals. Thus, CO-iM is a robust model to decipher neuropathogenesis, neurological disorders, and viral infections of brain cells in a 3D culture system.

An In Vitro Model of Blood-Brain Barrier for Studies on HIV Neuroinflammation and CNS Antibody Penetration.

The blood-brain barrier (BBB) is one of several barriers between the brain and the peripheral blood system to maintain homeostasis. Understanding the interactions between infectious agents such as human immunodeficiency virus type 1 (HIV-1), which are capable of traversing the BBB and causing neuroinflammation requires modeling an authentic BBB in vitro. Such an in vitro BBB model also helps develop means of targeting viruses that reside in the brain via natural immune effectors such as antibodies. The BBB consists of human brain microvascular endothelial cells (HBMECs), astrocytes, and pericytes. Here we report in vitro methods to establish a dual-cell BBB model consisting of primary HBMECs and primary astrocytes to measure the integrity of the BBB and antibody penetration of the BBB, as well as a method to establish a single cell BBB model to study the impact of HIV-1 infected medium on the integrity of such a BBB.

Bulk IgG glycosylation predicts COVID-19 severity and vaccine antibody response.

Although vaccination efforts have expanded, there are still gaps in our understanding surrounding the immune response to SARS-CoV-2. Measuring IgG Fc glycosylation provides insight into an infected individual's inflammatory state, among other functions. We set out to interrogate bulk IgG glycosylation changes from SARS-CoV-2 infection and vaccination, using plasma from mild or hospitalized COVID-19 patients, and from vaccinated individuals. Inflammatory glycans are elevated in hospitalized COVID-19 patients and increase over time, while mild patients have anti-inflammatory glycans that increase over time, including increased sialic acid correlating with RBD antibody levels. Vaccinated individuals with low RBD antibody levels and low neutralization have the same IgG glycan traits as hospitalized COVID-19 patients. In addition, a small vaccinated cohort reveals a decrease in inflammatory glycans associated with peak IgG concentrations and neutralization. This report characterizes the bulk IgG glycome associated with COVID-19 severity and vaccine responsiveness and can help guide future studies into SARS-CoV-2 protective immunity.

Divergent Antibody Repertoires Found for Omicron versus Wuhan SARS-CoV-2 Strains Using Ig-MS.

SARS-CoV-2 Omicron (B.1.1.529) and its subvariants are currently the most common variants of concern worldwide, featuring numerous mutations in the spike protein and elsewhere that collectively make Omicron variants more transmissible and more resistant to antibody-mediated neutralization provided by vaccination, previous infections, and monoclonal antibody therapies than their predecessors. We recently reported the creation and characterization of Ig-MS, a new mass spectrometry-based serology platform that can define the repertoire of antibodies against an antigen of interest at single proteoform resolution. Here, we applied Ig-MS to investigate the evolution of plasma antibody repertoires against the receptor-binding domain (RBD) of SARS-CoV-2 in response to the booster shot and natural viral infection. We also assessed the capacity for antibody repertoires generated in response to vaccination and/or infection with the Omicron variant to bind to both Wuhan- and Omicron-RBDs. Our results show that (1) the booster increases antibody titers against both Wuhan- and Omicron- RBDs and elicits an Omicron-specific response and (2) vaccination and infection act synergistically in generating anti-RBD antibody repertoires able to bind both Wuhan- and Omicron-RBDs with variant-specific antibodies.

De novo emergence of SARS-CoV-2 spike mutations in immunosuppressed patients.

BACKGROUND: The continuing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with decreased susceptibility to neutralizing antibodies is of clinical importance. Several spike mutations associated with immune escape have evolved independently in association with different variants of concern (VOCs). How and when these mutations arise is still unclear. We hypothesized that such mutations might arise in the context of persistent viral replication in immunosuppressed hosts. METHODS: Nasopharyngeal specimens were collected longitudinally from two immunosuppressed patients with persistent SARS-CoV-2 infection. Plasma was collected from these same patients late in disease course. SARS-CoV-2 whole genome sequencing was performed to assess the emergence and frequency of mutations over time. Select Spike mutations were assessed for their impact on viral entry and antibody neutralization in vitro. RESULTS: Our sequencing results revealed the intrahost emergence of spike mutations that are associated with circulating VOCs in both immunosuppressed patients (del241-243 and E484Q in one patient, and E484K in the other). These mutations decreased antibody-mediated neutralization of pseudotyped virus particles in cell culture, but also decreased efficiency of spike-mediated cell entry. CONCLUSIONS: These observations demonstrate the de novo emergence of SARS-CoV-2 spike mutations with enhanced immune evasion in immunosuppressed patients with persistent infection. These data suggest one potential mechanism for the evolution of VOCs and emphasize the importance of continued efforts to develop antiviral drugs for suppression of viral replication in hospitalized settings.

Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19.

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes on the same analyte remain limited. This report describes the development and optimization of a multiplexed immunobead assay for serological testing of common immunoglobulin isotypes (e.g., IgA, IgM, and IgG) associated with an immune response to SARS-CoV-2 infection or vaccination. Assays were accomplished using a flow-based, multiplex fluorescent reader with dual-channel capability. Optimizations focused on analyte capture time, detection antibody concentration, and detection antibody incubation time. Analytical assay performance characteristics (e.g., assay range (including lower and upper limits of quantitation); and intra- and inter-assay precision) were established for either IgG/IgM or IgA/IgM serotype combination in tandem using the 'dual channel' mode. Analyte capture times of 30 min for IgG, 60 min for IgM, and 120 min for IgA were suitable for most applications, providing a balance of assay performance and throughput. Optimal detection antibody incubations at 4 µg/mL for 30 min was observed and are recommended for general applications, given the overall excellent precision (percent coefficient of variance (%CV) ≤ 20%) and sensitivity values observed. The dynamic range for the IgG isotype spanned several orders of magnitude for each assay (Spike S1, Nucleocapsid, and Membrane glycoproteins), which supports robust titer evaluations at a 1:500 dilution factor for clinical applications. Finally, the optimized protocol was applied to monitoring Spike S1 seroconversion for subjects (n = 4) that completed a SARS-CoV-2 vaccine regimen. Within this cohort, Spike S1 IgG levels were observed to reach maximum titers at 14 days following second dose administration, at a much higher (~40-fold) signal intensity than either IgM or IgA isotypes. Interestingly, we observed highly variable Spike S1 IgG titer decay rates that were largely subject-dependent were observed, which will be the topic of future studies.

Tracking fluorescently labeled IL-15 and anti-PD-1 in the tumor microenvironment and draining lymph nodes.

Understanding the dynamics of the tumor microenvironment (TME) has become vital in discovering new targets for effective immunotherapies and enhancing current treatments. However, localization and distribution of immune cells and treatment biomolecules are poorly characterized to date. In this study, a murine Luminal B mammary adenocarcinoma model received a combinatorial treatment of fluorescently labeled anti-PD-1-Cy3 and IL-15 complex-Cy5 injected interperitoneally and intratumorally, respectively. Fluorescent labeling allowed for the visualization of the distribution of IL-15 complexes and anti-PD-1, as well as their localization to immune cells in the TME and tumor-draining lymph node. Using fluorescent microscopy and light sheet microscopy of whole-clarified tumors and draining lymph nodes, the localization of IL-15 complexes was found to be distributed around the periphery of the tumor at 4 h post injection and medially located at the center of the tumor at 24 h post injection, corresponding with high densities of CD8 cells in the tumor present at 48 h and 72 h post injection. Anti-PD-1 was distributed around the perimeter of the tumor and colocalized to IL-15 in the draining lymph nodes 24 h post injection. Colocalization of IL-15 was also established with NK cells, CD8+ T cells, and macrophages. This study develops a novel method to spatiotemporally track fluorescently labeled immunotherapeutic biomolecules in vivo, with implications for optimizing and further understanding the pharmacokinetics of clinical immunotherapies.

Multiple expansions of globally uncommon SARS-CoV-2 lineages in Nigeria.

Disparities in SARS-CoV-2 genomic surveillance have limited our understanding of the viral population dynamics and may delay identification of globally important variants. Despite being the most populated country in Africa, Nigeria has remained critically under sampled. Here, we report sequences from 378 SARS-CoV-2 isolates collected in Oyo State, Nigeria between July 2020 and August 2021. In early 2021, most isolates belonged to the Alpha "variant of concern" (VOC) or the Eta lineage. Eta outcompeted Alpha in Nigeria and across West Africa, persisting in the region even after expansion of an otherwise rare Delta sub-lineage. Spike protein from the Eta variant conferred increased infectivity and decreased neutralization by convalescent sera in vitro. Phylodynamic reconstructions suggest that Eta originated in West Africa before spreading globally and represented a VOC in early 2021. These results demonstrate a distinct distribution of SARS-CoV-2 lineages in Nigeria, and emphasize the need for improved genomic surveillance worldwide.

Next-Generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multiparametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We applied Ig-MS to plasma from subjects with severe and mild COVID-19 and immunized subjects after two vaccine doses, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, that could use other antigens of interest to gauge immune responses to vaccination, pathogens, or autoimmune disorders.

HIV in the Brain: Identifying Viral Reservoirs and Addressing the Challenges of an HIV Cure.

Advances in antiretroviral therapy have prolonged the life of people living with HIV and diminished the level of virus in these individuals. Yet, HIV quickly rebounds after disruption and/or cessation of treatment due to significant cellular and anatomical reservoirs for HIV, which underscores the challenge for HIV cure strategies. The central nervous system (CNS), in particular, is seeded with HIV within 1-2 weeks of infection and is a reservoir for HIV. In this review, we address the paradigm of HIV reservoirs in the CNS and the relevant cell types, including astrocytes and microglia, that have been shown to harbor viral infection even with antiretroviral treatment. In particular, we focus on developmental aspects of astrocytes and microglia that lead to their susceptibility to infection, and how HIV infection propagates among these cells. We also address challenges of measuring the HIV latent reservoir, advances in viral detection assays, and how curative strategies have evolved in regard to the CNS reservoir. Current curative strategies still require optimization to reduce or eliminate the HIV CNS reservoir, and may also contribute to levels of neuroinflammation that lead to cognitive decline. With this in mind, the latent HIV reservoir in the brain should remain a prominent focus when assessing treatment options and overall viral burden in the clinic, especially in the context of HIV-associated neurocognitive disorders (HAND).

Next-generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS , a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multi-parametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We apply Ig-MS to plasma from subjects with severe & mild COVID-19, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, with compatibility to any recombinant antigen to gauge our immune responses to vaccination, pathogens, or autoimmune disorders.

Anatomic Distribution of Intravenously Injected IgG Takes Approximately 1 Week to Achieve Stratum Corneum Saturation in Vaginal Tissues.

i.v. injected Abs have demonstrated protection against simian HIV infection in rhesus macaques, paving the way for the Antibody Mediated Prevention trial in which at-risk individuals for HIV received an i.v. infusion of the HIV broadly neutralizing Ab VRC01. However, the time needed for these Abs to fully distribute and elicit protection at mucosal sites is still unknown. In this study, we interrogate how long it takes for Abs to achieve peak anatomical levels at the vaginal surface following i.v. injection. Fluorescently labeled VRC01 and/or Gamunex-C were i.v. injected into 24 female rhesus macaques (Macaca mulatta) with vaginal tissues and plasma acquired up to 2 wk postinjection. We found that Ab delivery to the vaginal mucosa occurs in two phases. The first phase involves delivery to the submucosa, occurring within 24 h and persisting beyond 1 wk. The second phase is the delivery through the stratified squamous epithelium, needing ∼1 wk to saturate the stratum corneum. This study has important implications for the efficacy of immunoprophylaxis targeting pathogens at the mucosa.

PET/CT targeted tissue sampling reveals virus specific dIgA can alter the distribution and localization of HIV after rectal exposure.

Human immunodeficiency virus (HIV) vaccines have not been successful in clinical trials. Dimeric IgA (dIgA) in the form of secretory IgA is the most abundant antibody class in mucosal tissues, making dIgA a prime candidate for potential HIV vaccines. We coupled Positron Emission Tomography (PET) imaging and fluorescent microscopy of 64Cu-labeled, photoactivatable-GFP HIV (PA-GFP-BaL) and fluorescently labeled dIgA to determine how dIgA antibodies influence virus interaction with mucosal barriers and viral penetration in colorectal tissue. Our results show that HIV virions rapidly disseminate throughout the colon two hours after exposure. The presence of dIgA resulted in an increase in virions and penetration depth in the transverse colon. Moreover, virions were found in the mesenteric lymph nodes two hours after viral exposure, and the presence of dIgA led to an increase in virions in mesenteric lymph nodes. Taken together, these technologies enable in vivo and in situ visualization of antibody-virus interactions and detailed investigations of early events in HIV infection.

Coincident rapid expansion of two SARS-CoV-2 lineages with enhanced infectivity in Nigeria.

The emergence of new SARS-CoV-2 variants with enhanced transmissibility or decreased susceptibility to immune responses is a major threat to global efforts to end the coronavirus disease 2019 (COVID-19) pandemic. Disparities in viral genomic surveillance capabilities and efforts have resulted in gaps in our understanding of the viral population dynamics across the globe. Nigeria, despite having the largest population of any nation in Africa, has had relatively little SARS-CoV-2 sequence data made publicly available. Here we report the whole-genome sequences of 74 SARS-CoV-2 isolates collected from individuals in Oyo State, Nigeria in January 2021. Most isolates belonged to either the B.1.1.7 Alpha "variant of concern" or the B.1.525 Eta lineage, which is currently considered a "variant of interest" containing multiple spike protein mutations previously associated with enhanced transmissibility and possible immune escape. Nigeria has the highest reported frequency of the B.1.525 lineage globally with phylogenetic characteristics consistent with a recent monophyletic origin and rapid expansion. Spike protein from the B.1.525 lineage displayed both increased infectivity and decreased neutralization by convalescent sera compared to Spike proteins from other clades. These results, along with indications that the virus is outpacing the B.1.1.7 lineage in Nigeria, suggest that the B.1.525 lineage represents another "variant of concern" and further underline the importance of genomic surveillance in undersampled regions across the globe.

A MUC16 IgG Binding Activity Selects for a Restricted Subset of IgG Enriched for Certain Simian Immunodeficiency Virus Epitope Specificities.

We have recently shown that MUC16, a component of the glycocalyx of some mucosal barriers, has elevated binding to the G0 glycoform of the Fc portion of IgG. Therefore, IgG from patients chronically infected with human immunodeficiency virus (HIV), who typically exhibit increased amounts of G0 glycoforms, showed increased MUC16 binding compared to uninfected controls. Using the rhesus macaque simian immunodeficiency virus SIVmac251 model, we can compare plasma antibodies before and after chronic infection. We find increased binding of IgG to MUC16 after chronic SIV infection. Antibodies isolated for tight association with MUC16 (MUC16-eluted antibodies) show reduced FcγR engagement and antibody-dependent cellular cytotoxicity (ADCC) activity. The glycosylation profile of these IgGs was consistent with a decrease in FcγR engagement and subsequent ADCC effector function, as they contain a decrease in afucosylated bisecting glycoforms that preferentially bind FcγRs. Testing of the SIV antigen specificity of IgG from SIV-infected macaques revealed that the MUC16-eluted antibodies were enriched for certain specific epitopes, including regions of gp41 and gp120. This enrichment of specific antigen responses for fucosylated bisecting glycoforms and the subsequent association with MUC16 suggests that the immune response has the potential to direct specific epitope responses to localize to the glycocalyx through interaction with this specific mucin.IMPORTANCE Understanding how antibodies are distributed in the mucosal environment is valuable for developing a vaccine to block HIV infection. Here, we study an IgG binding activity in MUC16, potentially representing a new IgG effector function that would concentrate certain antibodies within the glycocalyx to trap pathogens before they can reach the underlying columnar epithelial barriers. These studies reveal that rhesus macaque IgG responses during chronic SIV infection generate increased antibodies that bind MUC16, and interestingly, these MUC16-tethered antibodies are enriched for binding to certain antigens. Therefore, it may be possible to direct HIV vaccine-generated responses to associate with MUC16 and enhance the antibody's ability to mediate immune exclusion by trapping virions within the glycocalyx and preventing the virus from reaching immune target cells within the mucosa. This concept will ultimately have to be tested in the rhesus macaque model, which is shown here to have MUC16-targeted antigen responses.

Long-term direct visualization of passively transferred fluorophore-conjugated antibodies.

The use of therapeutic antibodies, delivered by intravenous (IV) instillation, is a rapidly expanding area of biomedical treatment for a variety of conditions. However, little is known about how the antibodies are anatomically distributed following infusion and the underlying mechanism mediating therapeutic antibody distribution to specific anatomical sites remains to be elucidated. Current efforts utilize low resolution and sensitivity methods such as ELISA and indirect labeling imaging techniques, which often leads to high background and difficulty in assessing biodistribution. Here, using the in vivo non-human primate model, we demonstrate that it is possible to utilize the fluorophores Cy5 and Cy3 directly conjugated to antibodies for direct visualization and quantification of passively transferred antibodies in plasma, tissue, and in mucosal secretions. Antibodies were formulated with 1-2 fluorophores per antibody to minimally influence antibody function. Fluorophore conjugated Gamunex-C (pooled human IgG) were tested for binding to protein A, via surface plasmon resonance, and showed similar levels of binding when compared to unlabeled Gamunex-C. In order to assess the effect fluorophore labeling has on turnover and localization, rhesus macaques were IV infused with either labeled or unlabeled Gamunex-C. Plasma, vaginal Weck-Cel fluid, cervicovaginal mucus, and vaginal/rectal tissue biopsies were collected up to 8weeks. Similar turnover and biodistribution was observed between labeled and unlabeled antibodies, showing that the labeling process did not have an obvious deleterious effect on localization or turnover. Cy5 and Cy3 labeled antibodies were readily detected in the same pattern regardless of fluorophore. Tissue distribution was measured in macaque vaginal and rectal biopsies. The labeled antibody in macaque biopsies was found to have similar biodistribution pattern to endogenous antibodies in macaque and human tissues. In the vaginal and rectal mucosa, endogenous and infused antibodies were found primarily within the lamina propria. In the mucosal squamous epithelium of the vaginal vault, significant antibody was also observed in a striated pattern in the superficial, nonviable, stratum corneum. Endogenous antibody distribution in both human and macaque squamous tissues exhibited a similar pattern as seen with the labeled and unlabeled antibodies. This proof-of-principle study reveals that the labeled antibody is stable and physiologically similar relative to endogenous antibody setting the stage for future work to better understand the mechanisms of how antibodies reach unique anatomical sites. Direct visualization of fluorophore-conjugated antibodies following passive infusion can be utilized to assess the kinetics of biodistribution of infused antibodies and may be a useful approach to monitor and predict efficacy of therapeutic antibodies.

Regression of massive cardiac rhabdomyoma on everolimus therapy.

Cardiac rhabdomyoma is the primary feature of the genetic disease, tuberous sclerosis complex, the most common cardiac tumor diagnosed in neonates and infants. Spontaneous regression is observed in most cases, but these tumors may cause hemodynamic instability, arrhythmias or other complications. We describe the case of a critically ill neonate, resuscitated after cardiac arrest secondary to massive locally invasive cardiac rhabdomyoma, who was successfully treated with everolimus (mammalian target of rapamycin [mTOR] inhibitor). Rapid tumor regression was observed on echocardiography, but it was unlikely that it was confounded by the natural disease course of regression. The presented case suggests that mTOR inhibitors may play a significant role in the treatment of large cardiac rhabdomyomas in critically ill neonates.

An Ubiquitin-like Motif in ASK1 Mediates its Association with and Inhibition of the Proteasome.

Linear polyubiquitin is processed at LRLRGG sequences by deubiquitinating enzymes to make free monomeric ubiquitin. This LRLRGG ubiquitin-like motif is found in a limited number of mammalian non-ubiquitin proteins, including the MAP3K Apoptosis Signal-Regulating Kinase-1 (ASK1), which activates MAPK signaling pathways. The c-terminus of ASK1 binds to the 19S cap of the proteasome allowing ASK1 to phosphorylate and inhibit proteasomal activity. We investigated whether the ubiquitin-like sequence in the c-terminus of ASK1 mediates its association with and inhibition of the proteasome. To test this we generated ASK1 with substitutions or deletions in this ubiquitin-like domain and examined the activation of cellular signaling and the association of ASK1 with the 19S cap of the proteasome. We show that ASK1 mutants have reduced association with the 19S cap of the proteasome, reduced capacity to inhibit the proteasome, and diminished ability to inhibit TNF-induced NF-κB activation. Mutant forms of ASK1 also had reduced capacity to activate JNK signaling, suggesting that the ubiquitin-like motif in ASK1 is also important for coordinating the balance between JNK and NF-κB signaling. Together these results demonstrate that the ubiquitin-like sequence of ASK1 is important for binding to and inhibition of the proteasome, and for the coordinated activation of cellular NF-κB and JNK signaling.

TNFAIP3 maintains intestinal barrier function and supports epithelial cell tight junctions.

Tight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3(-/-) mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3(-/-) mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.