Intestinal permeability and inflammatory features of juvenile age correlate with the eventual systemic autoimmunity in lupus-prone female SWR × NZB F1 (SNF1) mice.

The incidence of systemic lupus erythematosus (SLE) is about nine times higher in women than in men, and the underlying mechanisms that contribute to this gender bias are not fully understood. Previously, using lupus-prone (SWR × NZB)F1 (SNF1) mice, we have shown that the intestinal immune system could play a role in the initiation and progression of disease in SLE, and depletion of gut microbiota produces more pronounced disease protection in females than in males. Here, we show that the gut permeability features of lupus-prone female SNF1 mice at juvenile ages directly correlate with the expression levels of pro-inflammatory factors, faecal IgA abundance and nAg reactivity and the eventual systemic autoantibody levels and proteinuria onset. Furthermore, we observed that the disease protection achieved in female SNF1 mice upon depletion of gut microbiota correlates with the diminished gut inflammatory protein levels, intestinal permeability and circulating microbial DNA levels. However, faecal microbiota transplant from juvenile male and females did not result in modulation of gut inflammatory features or permeability. Overall, these observations suggest that the early onset of intestinal inflammation, systemic autoantibody production and clinical stage disease in lupus-prone females is linked to higher gut permeability in them starting at as early as juvenile age. While the higher gut permeability in juvenile lupus-prone females is dependent on the presence of gut microbes, it appears to be independent of the composition of gut microbiota.

Combination of Autophagy Selective Therapeutics With Doxil: An Assessment of Pathological Toxicity.

Background: Combination therapy of targeted drugs in cancer treatment is a field in constant flux, with research balancing side effects with efficacy. Efficacy from combination therapy is improved either through synthetic lethality or through prevention of recurrent clones. Previous research has shown (hydroxy-)chloroquine is insufficient to disrupt autophagy in tumors. Hence, either combinations or novel autophagy agents are desired. In vivo studies of ovarian cancer have revealed that chloroquine can be combined with up to four other autophagy drugs to suppress ovarian cancer growth. While cancer efficacy is now established for the autophagy drug combination, it is unclear what toxicities may require monitoring in human trials. Additive toxicity with chemotherapy is also unknown. Methods: To address toxicity in more depth than previous weight-monitoring studies, biochemical and histopathology studies were performed. Mouse groups were treated with autophagy drugs for 2 weeks, with or without the chemotherapy Doxil. After the last dose, mice were processed for blood biochemistry, white blood cell markers, and histopathology. Results: Data from a comprehensive blood biochemistry panel, flow cytometric measurements of blood cell markers, and histopathology are herein reported. While Doxil presented clear bone marrow and immunologic toxicity, autophagy drugs were overall less toxic and more variable in their presentation of potential toxicities. Only minor additive effects of autophagy drugs with Doxil were observed. Conclusion: Combinations of autophagy drugs may be considered for therapy in human oncology trials, with possible side effects to monitor informed by these murine pre-clinical data.

Activation of T cell checkpoint pathways during ß-cell antigen presentation by engineered dendritic cells promotes protection from type 1 diabetes.

Defective immune regulation has been recognized in type 1 diabetes (T1D). Immune regulatory T cell check-point receptors, which are generally upregulated on activated T cells, have been the molecules of attention as therapeutic targets for enhancing immune response in tumour therapy. Here, we show that pancreatic ß-cell antigen (BcAg) presentation by engineered tolerogenic dendritic cells (tDCs) that express CTLA4 selective ligand (B7.1wa) or a combination of CTLA4, PD1 and BTLA selective ligands (B7.1wa, PD-L1 and HVEM-CRD1 respectively; multiligand-DCs) causes an increase in regulatory cytokine and T cell (Treg) responses and suppression of the effector T cell function as compared with engineered control-DCs. Non-obese diabetic mice treated with BcAg-pulsed CTLA4-ligand-DCs and multiligand-DCs at pre-diabetic and early-hyperglycaemic stages showed significantly lower degree of insulitis, higher frequencies of insulin-positive islets, profound delay in and reversal of hyperglycaemia for a significant duration. Immune cells from the tDC-treated mice not only produced lower amounts of IFNγ and higher amounts of IL10 and TGFß1 upon BcAg challenge, but also failed to induce hyperglycaemia upon adoptive transfer. While both CTLA4-ligand-DCs and multiligand-DCs were effective in inducing tolerance, multiligand-DC treatment produced an overall higher suppressive effect on effector T cell function and disease outcome. These studies show that enhanced engagement of T cell checkpoint receptors during BcAg presentation can modulate T cell function and suppress autoimmunity and progression of the disease in T1D.

Gastrin producing syngeneic mesenchymal stem cells protect non-obese diabetic mice from type 1 diabetes.

Progressive destruction of pancreatic islet ß-cells by immune cells is a primary feature of type 1 diabetes (T1D) and therapies that can restore the functional ß-cell mass are needed to alleviate disease progression. Here, we report the use of mesenchymal stromal/stem cells (MSCs) for the production and delivery of Gastrin, a peptide hormone that is produced by intestinal cells and foetal islets and can increase ß-Cell mass, to promote protection from T1D. A single injection of syngeneic MSCs that were engineered to express Gastrin (Gastrin-MSCs) caused a significant delay in hyperglycaemia in non-obese diabetic (NOD) mice compared to engineered control-MSCs. Similar treatment of early-hyperglycaemic mice caused the restoration of euglycemia for a considerable duration, and these therapeutic effects were associated with the protection of, and/or higher frequencies of, insulin-producing islets and less severe insulitis. While the overall immune cell phenotype was not affected profoundly upon treatment using Gastrin-MSCs or upon in vitro culture, pancreatic lymph node cells from Gastrin-MSC treated mice, upon ex vivo challenge with self-antigen, showed a Th2 and Th17 bias, and diminished the diabetogenic property in NOD-Rag1 deficient mice suggesting a disease protective immune modulation under Gastrin-MSC treatment associated protection from hyperglycaemia. Overall, this study shows the potential of production and delivery of Gastrin in vivo, by MSCs, in protecting insulin-producing ß-cells and ameliorating the disease progression in T1D.

Loss of CPAP causes sustained EGFR signaling and epithelial-mesenchymal transition in oral cancer.

Higher epidermal growth factor receptor (EGFR) signaling can contribute to tumor metastasis and resistance to therapies in oral squamous cell carcinoma (OSCC). EGFR signaling can promote epithelial-mesenchymal transition (EMT) in OSCC. EMT is a process by which epithelial cells acquire invasive properties and it can contribute to tumor metastasis. Not only do the abnormal functions of microtubule and microtubule-organizing centers (MTOC) such as centrosomes lead to cancers, but also the malignant tissues are characterized by aberrant centriolar features and amplified centrosomes. Microtubule inhibition therapies increase the sensitivity to EGFR targeting drugs in various cancers. In this study, we show that the loss of expression of a microtubule/tubulin binding protein, centrosomal protein 4.1-associated protein (CPAP), which is critical for centriole biogenesis and normal functioning of the centrosome, caused an increase in the EGFR levels and its signaling and, enhanced the EMT features and invasiveness of OSCC cells. Further, depletion of CPAP enhanced the tumorigenicity of these cells in a xeno-transplant model. Importantly, CPAP loss-associated EMT features and invasiveness of multiple OSCC cells were attenuated upon depletion of EGFR in them. On the other hand, we found that CPAP protein levels were higher in EGF treated OSCC cells as well as in oral cancer tissues, suggesting that the frequently reported aberrant centriolar features of tumors are potentially a consequence, but not the cause, of tumor progression. Overall, our novel observations show that, in addition to its known indispensable role in centrosome biogenesis, CPAP also plays a vital role in suppressing tumorigenesis in OSCC by facilitating EGFR homeostasis.

Abundance and nuclear antigen reactivity of intestinal and fecal Immunoglobulin A in lupus-prone mice at younger ages correlate with the onset of eventual systemic autoimmunity.

Our recent studies, using (SWRxNZB)F1 (SNF1) mice, showed a potential contribution of the gut microbiota and pro-inflammatory immune responses of the gut mucosa to systemic autoimmunity in lupus. Here, using this mouse model, we determined the abundance and the nAg reactivity of IgA antibody produced in the intestine under lupus susceptibility. Intestinal lymphoid tissues from SNF1 mice, females particularly, showed significantly higher frequencies of nAg (dsDNA and nucleohistone) reactive IgA producing B cells compared to B6 females. Most importantly, younger age fecal IgA -abundance and -nAg reactivity of lupus-prone mice showed a positive correlation with eventual systemic autoimmunity and proteinuria onset. Depletion of gut microbiota in SNF1 mice resulted in the diminished production of IgA in the intestine and the nAg reactivity of these antibodies. Overall, these observations show that fecal IgA features, nuclear antigen reactivity particularly, at preclinical stages/in at-risk subjects could be predictive of autoimmune progression.

Engineered Dendritic Cell-Directed Concurrent Activation of Multiple T cell Inhibitory Pathways Induces Robust Immune Tolerance.

Inhibitory/repressor-receptors are upregulated significantly on activated T cells, and have been the molecules of attention as targets for inducing immune tolerance. Induction of effective antigen specific tolerance depends on concurrent engagement of the TCR and one or more of these inhibitory receptors. Here, we show, for the first time that dendritic cells (DCs) can be efficiently engineered to express multiple T cell inhibitory ligands, and enhanced engagement of T cell inhibitory receptors, upon antigen presentation, by these DCs can induce effective CD4+ T cell tolerance and suppress autoimmunity. Compared to control DCs, antigen presentation by DCs that ectopically express CTLA4, PD1 and BTLA selective ligands (B7.1wa, PD-L1, and HVEM-CRD1 respectively) individually (mono-ligand DCs) or in combination (multi-ligand DCs) causes an inhibition of CD4+ T cell proliferation and pro-inflammatory cytokine response, as well as increase in Foxp3+ Treg frequency and immune regulatory cytokine production. Administration of self-antigen (mouse thyroglobulin; mTg) loaded multi-ligand DCs caused hyporesponsiveness to mTg challenge, suppression of autoantibody production, and amelioration of experimental autoimmune thyroiditis. Overall, this study shows that engineered DC-directed enhanced concurrent activation of multiple T cell coinhibitory pathways is an effective way to induce self-antigen specific T cell tolerance to suppress ongoing autoimmunity.

Polysaccharide A-Dependent Opposing Effects of Mucosal and Systemic Exposures to Human Gut Commensal Bacteroides fragilis in Type 1 Diabetes.

Bacteroides fragilis (BF) is an integral component of the human colonic commensal microbiota. BF is also the most commonly isolated organism from clinical cases of intra-abdominal abscesses, suggesting its potential to induce proinflammatory responses upon accessing the systemic compartment. Hence, we examined the impact of mucosal and systemic exposures to BF on type 1 diabetes (T1D) incidence in NOD mice. The impact of intestinal exposure to BF under a chemically induced enhanced gut permeability condition, which permits microbial translocation, in T1D was also examined. While oral administration of heat-killed (HK) BF to prediabetic mice caused enhanced immune regulation and suppression of autoimmunity, resulting in delayed hyperglycemia, mice that received HK BF by intravenous injection showed rapid disease progression. Importantly, polysaccharide A-deficient BF failed to produce these opposing effects upon oral and systemic deliveries. Furthermore, BF-induced modulation of disease progression was observed in wild-type, but not TLR2-deficient, NOD mice. Interestingly, oral administration of BF under enhanced gut permeability conditions resulted in accelerated disease progression and rapid onset of hyperglycemia in NOD mice. Overall, these observations suggest that BF-like gut commensals can cause proinflammatory responses upon gaining access to the systemic compartment and contribute to T1D in at-risk subjects.