Single cell RNA-sequencing profiling to improve the translation between human IBD and in vivo models.

Inflammatory bowel disease (IBD) is an umbrella term for two conditions (Crohn's Disease and Ulcerative Colitis) that is characterized by chronic inflammation of the gastrointestinal tract. The use of pre-clinical animal models has been invaluable for the understanding of potential disease mechanisms. However, despite promising results of numerous therapeutics in mouse colitis models, many of these therapies did not show clinical benefits in patients with IBD. Single cell RNA-sequencing (scRNA-seq) has recently revolutionized our understanding of complex interactions between the immune system, stromal cells, and epithelial cells by mapping novel cell subpopulations and their remodeling during disease. This technology has not been widely applied to pre-clinical models of IBD. ScRNA-seq profiling of murine models may provide an opportunity to increase the translatability into the clinic, and to choose the most appropriate model to test hypotheses and novel therapeutics. In this review, we have summarized some of the key findings at the single cell transcriptomic level in IBD, how specific signatures have been functionally validated in vivo, and highlighted the similarities and differences between scRNA-seq findings in human IBD and experimental mouse models. In each section of this review, we highlight the importance of utilizing this technology to find the most suitable or translational models of IBD based on the cellular therapeutic target.

Tregs in Autoimmunity: Insights Into Intrinsic Brake Mechanism Driving Pathogenesis and Immune Homeostasis.

CD4+CD25highFoxp3+ regulatory T-cells (Tregs) are functionally characterized for their ability to suppress the activation of multiple immune cell types and are indispensable for maintaining immune homeostasis and tolerance. Disruption of this intrinsic brake system assessed by loss of suppressive capacity, cell numbers, and Foxp3 expression, leads to uncontrolled immune responses and tissue damage. The conversion of Tregs to a pathogenic pro-inflammatory phenotype is widely observed in immune mediated diseases. However, the molecular mechanisms that underpin the control of Treg stability and suppressive capacity are incompletely understood. This review summarizes the concepts of Treg cell stability and Treg cell plasticity highlighting underlying mechanisms including translational and epigenetic regulators that may enable translation to new therapeutic strategies. Our enhanced understanding of molecular mechanism controlling Tregs will have important implications into immune homeostasis and therapeutic potential for the treatment of immune-mediated diseases.

Endosomal Sequestration of TLR4 Antibody Induces Myeloid-Derived Suppressor Cells and Reverses Acute Type 1 Diabetes.

We previously showed that treating NOD mice with an agonistic monoclonal anti-TLR4/MD2 antibody (TLR4-Ab) reversed acute type 1 diabetes (T1D). Here, we show that TLR4-Ab reverses T1D by induction of myeloid-derived suppressor cells (MDSCs). Unbiased gene expression analysis after TLR4-Ab treatment demonstrated upregulation of genes associated with CD11b+Ly6G+ myeloid cells and downregulation of T-cell genes. Further RNA sequencing of purified, TLR4-Ab-treated CD11b+ cells showed significant upregulation of genes associated with bone marrow-derived CD11b+ cells and innate immune system genes. TLR4-Ab significantly increased percentages and numbers of CD11b+ cells. TLR4-Ab-induced CD11b+ cells, derived ex vivo from TLR4-Ab-treated mice, suppress T cells, and TLR4-Ab-conditioned bone marrow cells suppress acute T1D when transferred into acutely diabetic mice. Thus, the TLR4-Ab-induced CD11b+ cells, by the currently accepted definition, are MDSCs able to reverse T1D. To understand the TLR4-Ab mechanism, we compared TLR4-Ab with TLR4 agonist lipopolysaccharide (LPS), which cannot reverse T1D. TLR4-Ab remains sequestered at least 48 times longer than LPS within early endosomes, alters TLR4 signaling, and downregulates inflammatory genes and proteins, including nuclear factor-κB. TLR4-Ab in the endosome, therefore, induces a sustained, attenuated inflammatory response, providing an ideal "second signal" for the activation/maturation of MDSCs that can reverse acute T1D.

Enoxacin Up-Regulates MicroRNA Biogenesis and Down-Regulates Cytotoxic CD8 T-Cell Function in Autoimmune Cholangitis.

BACKGROUND AND AIMS: Primary biliary cholangitis (PBC) is a prototypical organ-specific autoimmune disease that is mediated by autoreactive T-cell attack and destruction of cholangiocytes. Despite the clear role of autoimmunity in PBC, immune-directed therapies have failed to halt PBC, including biologic therapies effective in other autoimmune diseases. MicroRNA (miRNA) dysregulation is implicated in the pathogenesis (PBC). In the dominant-negative TGF-ß receptor type II (dnTGFßRII) mouse model of PBC, autoreactive CD8 T cells play a major pathogenic role and demonstrate a striking pattern of miRNA down-regulation. Enoxacin is a small molecule fluoroquinolone that enhances miRNA biogenesis, partly by stabilizing the interaction of transactivation response RNA-binding protein with Argonaute (Ago) 2. APPROACH AND RESULTS: We hypothesized that correcting aberrant T-cell miRNA expression with enoxacin in dnTGFßRII mice could modulate autoreactive T-cell function and prevent PBC. Here, we show that liver-infiltrating dnTGFßRII CD8 T cells have significantly decreased levels of the miRNA biogenesis molecules prolyl 4-hydroxylase subunit alpha 1 (P4HA1) and Ago2 along with significantly increased levels of granzyme B and perforin. Enoxacin treatment significantly up-regulated miRNAs in dnTGFßRII CD8 T cells and effectively treated autoimmune cholangitis in dnTGFßRII mice. Enoxacin treatment directly altered T cells both ex vivo and in vitro, resulting in altered memory subset numbers, decreased proliferation, and decreased interferon-γ production. Enoxacin significantly decreased CD8 T-cell expression of the transcription factor, Runx3, and significantly decreased perforin expression at both the mRNA and protein levels. CONCLUSIONS: Enoxacin increases miRNA expression in dnTGFßRII CD8 T cells, reduces CD8 T-cell pathogenicity, and effectively halted progression of autoimmune biliary disease. Targeting the miRNA pathway is a therapeutic approach to autoimmunity that corrects pathological miRNA abnormalities in autoreactive T cells.

Exploiting CD22 To Selectively Tolerize Autoantibody Producing B-Cells in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects the synovial joints and can lead to bone erosion and cartilage damage. One hallmark of RA is anticitrullinated protein autoantibodies (ACPA) and memory citrulline-specific B-cells, which have been implicated in RA pathogenesis. While depletion of B-cells with Rituximab improves clinical responses in RA patients, this treatment strategy leaves patients susceptible to infections. Here we use of Siglec-engaging Tolerance-inducing Antigenic Liposomes (STALs) to selectively target the citrulline-specific B-cells. ACPA production from purified human RA patients' B-cells in vitro was achieved through a set of stimulation conditions, which includes the following: BAFF, anti-CD40, IL-21, and LPS. In vivo generation of citrulline specific B-cells and ACPA production was accomplished by antigenic liposomes consisting of monophosphoryl lipid A (MPLA) and a cyclic citrullinated peptide (CCP) administered to SJL/J mice. We show that STALs that codisplay a high affinity CD22 glycan ligand and synthetic citrullinated antigen (CCP STALs) can prevent ACPA production from RA patients' memory B-cells in vitro. These CCP STALs were also effective in inducing tolerance to citrullinated antigens in SJL/J mice. The results demonstrate that tolerization of the B-cells responsible for ACPA can be achieved by exploiting the inhibitory receptor CD22 with high-affinity glycan ligands. Such a treatment strategy could be beneficial in the treatment of RA.

Antigenic Liposomes for Generation of Disease-specific Antibodies.

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a naïve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Human CD22 Inhibits Murine B Cell Receptor Activation in a Human CD22 Transgenic Mouse Model.

CD22, a sialic acid-binding Ig-type lectin (Siglec) family member, is an inhibitory coreceptor of the BCR with established roles in health and disease. The restricted expression pattern of CD22 on B cells and most B cell lymphomas has made CD22 a therapeutic target for B cell-mediated diseases. Models to better understand how in vivo targeting of CD22 translates to human disease are needed. In this article, we report the development of a transgenic mouse expressing human CD22 (hCD22) in B cells and assess its ability to functionally substitute for murine CD22 (mCD22) for regulation of BCR signaling, Ab responses, homing, and tolerance. Expression of hCD22 on transgenic murine B cells is comparable to expression on human primary B cells, and it colocalizes with mCD22 on the cell surface. Murine B cells expressing only hCD22 have identical calcium (Ca2+) flux responses to anti-IgM as mCD22-expressing wild-type B cells. Furthermore, hCD22 transgenic mice on an mCD22-/- background have restored levels of marginal zone B cells and Ab responses compared with deficiencies observed in CD22-/- mice. Consistent with these observations, hCD22 transgenic mice develop normal humoral responses in a peanut allergy oral sensitization model. Homing of B cells to Peyer's patches was partially rescued by expression of hCD22 compared with CD22-/- B cells, although not to wild-type levels. Notably, Siglec-engaging antigenic liposomes formulated with an hCD22 ligand were shown to prevent B cell activation, increase cell death, and induce tolerance in vivo. This hCD22 transgenic mouse will be a valuable model for investigating the function of hCD22 and preclinical studies targeting hCD22.

CD137 Plays Both Pathogenic and Protective Roles in Type 1 Diabetes Development in NOD Mice.

We previously reported that CD137 (encoded by Tnfrsf9) deficiency suppressed type 1 diabetes (T1D) progression in NOD mice. We also demonstrated that soluble CD137 produced by regulatory T cells contributed to their autoimmune-suppressive function in this model. These results suggest that CD137 can either promote or suppress T1D development in NOD mice depending on where it is expressed. In this study, we show that NOD.Tnfrsf9-/- CD8 T cells had significantly reduced diabetogenic capacity, whereas absence of CD137 in non-T and non-B cells had a limited impact on T1D progression. In contrast, NOD.Tnfrsf9-/- CD4 T cells highly promoted T1D development. We further demonstrated that CD137 was important for the accumulation of ß cell-autoreactive CD8 T cells but was dispensable for their activation in pancreatic lymph nodes. The frequency of islet-infiltrating CD8 T cells was reduced in NOD.Tnfrsf9-/- mice in part because of their decreased proliferation. Furthermore, CD137 deficiency did not suppress T1D development in NOD mice expressing the transgenic NY8.3 CD8 TCR. This suggests that increased precursor frequency of ß cell-autoreactive CD8 T cells in NY8.3 mice obviated a role for CD137 in diabetogenesis. Finally, blocking CD137-CD137 ligand interaction significantly delayed T1D onset in NOD mice. Collectively, our results indicate that one important diabetogenic function of CD137 is to promote the expansion and accumulation of ß cell-autoreactive CD8 T cells, and in the absence of CD137 or its interaction with CD137 ligand, T1D progression is suppressed.

Reversal of New-Onset Type 1 Diabetes With an Agonistic TLR4/MD-2 Monoclonal Antibody.

Type 1 diabetes (T1D) is currently an incurable disease, characterized by a silent prodromal phase followed by an acute clinical phase, reflecting progressive autoimmune destruction of insulin-producing pancreatic ß-cells. Autoreactive T cells play a major role in ß-cell destruction, but innate immune cell cytokines and costimulatory molecules critically affect T-cell functional status. We show that an agonistic monoclonal antibody to TLR4/MD-2 (TLR4-Ab) reverses new-onset diabetes in a high percentage of NOD mice. TLR4-Ab induces antigen-presenting cell (APC) tolerance in vitro and in vivo, resulting in an altered cytokine profile, decreased costimulatory molecule expression, and decreased T-cell proliferation in APC:T-cell assays. TLR4-Ab treatment increases T-regulatory cell (Treg) numbers in both the periphery and the pancreatic islet, predominantly expanding the Helios(+)Nrp-1(+)Foxp3(+) Treg subset. TLR4-Ab treatment in the absence of B cells in NOD.scid mice prevents subsequent T cell-mediated disease, further suggesting a major role for APC tolerization in disease protection. Specific stimulation of the innate immune system through TLR4/MD-2, therefore, can restore tolerance in the aberrant adaptive immune system and reverse new-onset T1D, suggesting a novel immunological approach to treatment of T1D in humans.

Recombinant soluble CD137 prevents type one diabetes in nonobese diabetic mice.

Nonobese diabetic (NOD) mice are genetically programmed to spontaneously develop type one diabetes (T1D). Multiple Insulin dependent diabetes (Idd) genetic loci have been identified but their functional effects are mostly poorly understood. TnfsfR9, expressing the protein product CD137, is a strong candidate gene in the Idd9.3 locus, and NOD.B10 Idd9.3 mice are significantly protected from type one diabetes (T1D). We previously showed that nonobese diabetic (NOD) mice have a deficiency in the numbers of CD137(pos) T regulatory cells, that CD137(pos) Tregs are the source of soluble CD137 (sCD137), and that NOD mice have low serum levels of sCD137. To test the hypothesis that correcting low levels of sCD137 could affect the disease, we constructed a lentiviral vector producing recombinant sCD137; this physiologic sCD137 is glycosylated and exists primarily as a dimer. NOD mice treated with the recombinant sCD137 are protected from developing T1D. Insulitis is significantly decreased, but not eliminated in the sCD137 treated mice, however insulin producing pancreatic beta cells are preserved despite residual insulitis. To begin to understand the protective immune mechanisms of sCD137, we tested sCD137 in vitro. It was previously suggested that sCD137 simply blocked the interaction between CD137 (on T cells) and CD137 ligand (on antigen presenting cells (APCs)). Here however, we use an APC independent assay and demonstrate that sCD137 can actively suppress highly purified CD4 T cells in a CD137L dependent fashion. These results support the hypothesis that sCD137 acts in a negative feedback loop to actively suppress over-zealous immune responses, and that it can be used clinically to suppress autoimmunity. sCD137 is an important Treg derived natural immunosuppressive molecule that regulates effector T cells to avert diabetes in vivo.

Disruption of Staphylococcus epidermidis biofilm formation using a targeted cationic peptide.

This study reports the use of a targeted cationic peptide with the ability to disrupt Staphylococcus epidermidis biofilm formation. Complications due to nosocomial infections of implanted medical devices pose a significant health risk to patients, with Staphylococcus epidermidis often implicated in the case of blood-contacting biomaterials. S. epidermidis virulence relies mainly on its ability to form a biofilm, the main component of which is polysaccharide intercellular adhesin (PIA). We utilized the synthetic ß6-20 peptide, known to specifically bind S. epidermidis, in order to deliver a cationic polylysine peptide (G(3)K(6)) to the bacterial surface and disrupt the charge-charge interactions needed for PIA retention and biofilm stability. The effects of the ß6-20-G(3)K(6) peptide on biofilm formation were assessed using optical density, fluorescently labeled wheat germ agglutinin, nucleic acid stain (SYTO 9), and a metabolic assay (XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt). Biofilms formed in the presence of ß6-20-G(3)K(6) peptide (100 µM) resulted in a 37.9% reduction in PIA content and a 17.5% reduction of adherent bacteria relative to biofilms grown in the absence of peptide. These studies demonstrate the targeting ability of the ß6-20 peptide towards biomaterial-adherent S. epidermidis, and highlight the potential for disrupting the early stages of biofilm formation.