Multiple Sclerosis (MS) is an inflammatory disease of the Central Nervous System (CNS) that causes the demyelination of nerve cells and destroys oligodendrocytes, neurons and axons. Historically, MS has been thought of as a T cell-mediated autoimmune disease of CNS white matter. However, recent studies have identified gray matter lesions in MS patients, suggesting that CNS antigens other than myelin proteins may be involved during the MS disease process. We have recently found that T cells targeting astrocyte-specific antigens can drive unique aspects of inflammatory CNS autoimmunity, including the targeting of gray matter and white matter of the brain and inducing heterogeneous clinical disease courses. In addition to being a target of T cells, astrocytes play a critical role in propagating the inflammatory response within the CNS induced NF-κB signaling. Here, we will discuss the pathophysiology of CNS inflammation mediated by T cell—glial cell interactions and its contributions to CNS autoimmunity.
Enterohemorrhagic Escherichia coli produce ribotoxic Shiga toxins (Stx), which are responsible for kidney injury and development of hemolytic uremic syndrome. The endoplasmic reticulum (ER) stress response is hypothesized to induce apoptosis contributing to organ injury; however, this process has been described only in vitro. ER stress marker transcripts of spliced XBP1 (1.78-fold), HSP40 (4.45-fold) and CHOP (7.69-fold) were up-regulated early in kidneys of Stx2 challenged mice compared to saline controls. Anti-apoptotic Bcl2 decreased (−2.41-fold vs. saline) and pro-apoptotic DR5 increased (6.38-fold vs. saline) at later time points. Cytoprotective activated protein C (APC) reduced early CHOP expression (−3.3-fold vs. untreated), increased later Bcl2 expression (5.8-fold vs. untreated), and had early effects on survival but did not alter DR5 expression. Changes in kidney ER stress and apoptotic marker transcripts were observed in Stx2-producing C. rodentium challenged mice compared to mice infected with a non-toxigenic control strain. CHOP (4.14-fold) and DR5 (2.81-fold) were increased and Bcl2 (−1.65-fold) was decreased. APC reduced CHOP expression and increased Bcl2 expression, but did not alter mortality. These data indicate that Stx2 induces renal ER stress and apoptosis in murine models of Stx2-induced kidney injury, but decreasing these processes alone was not sufficient to alter survival outcome.
Hemolytic uremic syndrome (HUS) from enterohemorrhagic Escherichia coli infection is a leading cause of kidney failure in otherwise healthy U.S. children. The bacterial Shiga toxins (Stx) induce the characteristic coagulopathy of HUS, but the damage to toxin-receptor expressing cells and organ injury due to ischemia likely also releases inflammatory damage-associated molecular patterns (DAMPs), which may exacerbate injury along with the toxins. To examine this, human aortic and renal glomerular cell anti-coagulant and barrier functions were studied after in vitro challenge with Stx1, Stx2, and DAMPs. There was significant loss of surface anti-coagulant protein C pathway molecules, increased expression of pro-thrombotic PAR1 and reduced protein C activation capability by 15–27%. Histones nearly completely prevented the activated protein C protection of endothelial cells from thrombin-induced permeability. In mice, lethal Stx2 challenge elevated plasma HMGB1 (day 2, 321 ± 118%; p < 0.01) and extracellular histones (day 3, 158 ± 62%; p < 0.01). Mice colonized with Stx2-expressing Citrobacter rodentium developed increased HMGB1 (day 5, 155 ± 55%; p < 0.01) and histones (day 3, 378 ± 188%; p < 0.01). Anti-histone antibody reduced both DAMPs to baseline, but was not sufficient to improve survival outcome or kidney function. Together, these data suggest a potential role Stx to produce DAMPs, and DAMPs to produce endothelial injury and a pro-thrombotic environment.
Adaptive immunity is predicated on the ability of the T cell repertoire to have pre-existing specificity for the universe of potential pathogens. Recent findings suggest that TCR-self-pMHC interactions limit autoimmune responses while enhancing T cell response to foreign antigens. We review these findings here, placing them in context of the current understanding of how TCR-self-pMHC interactions regulate T cell activation thresholds, and suggest that TCR-self-pMHC interactions increase the efficiency of the T cell repertoire by giving a competitive advantage to peptide cross-reactive T cells. We propose that self-reactivity and peptide-cross-reactivity are controlled by particular CDR3 sequence motifs, which would allow thymic selection to contribute to solving the feat of broad pathogen-specificity by exporting T cells that are pre-screened by positive and negative selection for the ability to be ‘moderately’ peptide cross-reactive.
BACKGROUND: Intestinal barrier dysfunction exists when the epithelial tight-junctions are disrupted and permit translocation of bacterial lipopolysaccharides into the blood. The resulting endotoxemia cause systemic low-grade inflammation that contributes to the development of comorbidities associated with obesity like type 2 diabetes and NASH. Superabsorbent hydrogels based on the Gelesis platform technology are orally administered and synthesized from crosslinked modified cellulose. Gelesis100, the first in this class, demonstrated significant weight loss in patients with obesity and improvement in insulin sensitivity. We hypothesized that Gel-B with specific mechanical properties, designed to improve intestinal barrier function, would prevent translocation of small molecule into the blood after intestinal barrier injury. METHODS: Groups of 8 to 15 C57BI/6 male mice were administered 3% dextran sodium sulfate (DSS) in drinking water for a total of 6 days, and randomized to either Vehicle, Gel-B (0.5 to 4%) in their food from days 5 to 19. An active control group received anti-p40 IP q3 days on days 6-18. On day 19, all animals were fasted for 4 hours prior to FITC-Dextran dosing. Three hours post dosing, the mice were sacrificed, colon specimen fixed, and blood collected. The incidence of epithelial barrier breaks was assessed with E-cadherin immunohistochemistry. To verify the involvement of epithelial tight junctions, the expression of ZO-1 was assessed in C57BL/6J mice (5 per group) fed chow, for 4 weeks, with or without Gel-B. RESULTS: The level of FITC-Dextran in the serum of mice receiving DSS and vehicle (mean ± SEM, 7.1 ± 4.1) was 3.4 times higher than the naïve mice (2.1 ± 1.1) demonstrating a significant defect in the gut barrier function. In turn, the mice assigned to Gel-B 1% to 4% had meaningful reduction in FITC-Dextran serum levels compared to vehicle treated mice. The highest dose (4%) of Gel-B prevented spilling of dextran into the circulation with serum levels numerically lower than the naïve group with an intact gut barrier (1.4 ± 0.4 compared to 2.1 ± 1.1, respectively). Accordingly, a reduction in epithelial barrier breaks in distal colon samples was observed in the highest dose of Gel-B (0.58 ± 0.15) compared to vehicle treated animals (0.86 ± 0.1). Healthy mice treated with Gel-B (8%) showed increased expression of ZO-1, essential to tight junction assembly between epithelial cells. CONCLUSION: The present study demonstrated that treatment with Gel-B prevented the translocation of small molecule into the circulation indicating an improved intestinal barrier function. This effect may be explained by a decrease in gaps between the epithelial cells, and increased expression of tight junctions. These findings and future studies could provide new therapeutic approaches for diseases affected by a compromised intestinal barrier function such as obesity, diabetes and NASH. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of pre...
Mounting clinical and preclinical evidence demonstrates an important role for the intestinal microbiome in mediating efficacy of immune checkpoint inhibitor (ICI) antibody therapy across a number of tumor contexts. We sought to determine the effects of microbiome modulation on ICI therapy in a clinically recapitulative orthotopic murine lung cancer model. The microbiome of C57Bl/6NHsd mice was sterilized with antibiotic (ampicillin, streptomycin and colistin) for ten days, resulting in a 99.9% mean decrease in fecal aerobic and anaerobic bacterial load in comparison to naïve mice, handled under specific pathogen free (SPF) conditions. Luciferase-expressing murine Lewis lung carcinoma cells (LL/2-Red-FLuc) were surgically implanted into the left lung parenchyma of all animals. Animals were treated with locoregional radiotherapy (2x 9Gy fractions) targeted to the left lung. The gastrointestinal microbiome was reconstituted via oral gavage Q3D of ~1e09/dose commensal A. muciniphila (A. muc) and E. hirae (E. hir) over five doses or sterile saline as control and animals were randomized within commensal/saline treatment groups to equivalent mean tumor burden as measured by Lumina Series III In-Vivo Imaging System (IVIS). Animals were then administered anti-murine PD-1 or isotype control (0.25 mg) antibody (Ab) treatments Q3D over four doses via intraperitoneal injection. Tumor growth was monitored by IVIS over the course of the study, and ex-vivo IVIS was performed on lungs at endpoint (Day 12 post-tumor implantation). Tumor growth of microbiota non-reconstituted antibiotic-sterilized animals was only slightly inhibited by anti-PD-1 therapy. Animals administered commensal A. muc and E. hir and treatments also displayed slightly inhibited tumor growth kinetics, similar to those observed under saline/anti-PD-1 therapy. Fecal microbial sequencing and immunophenotypic analyses are ongoing. This study demonstrates the utility and ongoing development of a clinically recapitulative contextually accurate preclinical murine lung cancer model to assess the effects of specific microbiota in mediating the efficacy of anti-tumor immunotherapy. Citation Format: Benjamin G. Cuiffo, Caitlin S. Parello, Chelsea Ritchie, Nicholas Rivelli, Alexandra Kury, Sallyann Vu, Gavin Gagnon, Veronica Ritchie, Kasey Reardon, Catarina Costa, Samantha Rogers, Gregory D. Lyng, Stephen T. Sonis. Recolonizing microbiota may impact tumor response to PD-1 inhibition following antibiotic and radiotherapy treatment in a bioluminescent orthotopic model of murine lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1499.
Humanized immune system (HIS) mice - immunocompromised Nod.Cg-Prkdcscid Il2rgtm1Wjl/SzJ animals in which the immune system has been humanized by the engraftment of CD34+ hematopoietic stem cells (HSC), peripheral blood mononuclear cells (PBMC), or other human cells/tissues - represent an important tool in pre-clinical oncology research. Here we investigated several pre-conditioning regimens (Busulfan pre-treatment or total body irradiation (TBI)) to determine which would yield maximum human leukocyte engraftment and lineage diversity. Animals were pre-treated either one or two doses of Busulfan (25 mg/kg) or varying doses of TBI (1.75, 2.0, or 2.5 Gy). One to two days later, animals received an adoptive transfer of native hCD34+ HSCs. Engraftment was monitored by flow cytometry assessment of peripheral blood four weeks after adoptive-transfer, and every four weeks thereafter through experiment termination at Week 16. Upon termination, spleen and bone-marrow were assessed for various leukocyte populations by flow cytometry. Animals undergoing pre-conditioning displayed increased human CD45+ cell engraftment at all timepoints as compared to animals without pre-conditioning, with animals treated with two doses of Busulfan demonstrating the greatest human CD45+ engraftment at all timepoints and reaching statistical significance at endpoint (vs no pre-conditioning; p<0.05). Diversity of leukocyte cell types (B cells, T cells, NK cells, and other hCD45+) in the bone-marrow was similar across pre-treatment paradigms, however for some lineages, representation in the bone-marrow was significantly altered between pre-conditioning type. The absolute number of T or B cells were increased in TBI and Busulfan treated groups, respectively, whereas NK cells as a percentage of singlets were increased in TBI treated groups. The diversity of leukocyte cell types in the spleen was likewise similar across groups, with B cells dominating. These data provide important insights into the utility of various pre-conditioning regimens for the humanization of NSG mice for pre-clinical oncology research, and demonstrate that the optimum pre-conditioning regimens should be selected based on treatment target. Citation Format: Caitlin S. Parello, Benjamin G. Cuiffo, Alexandria Kury, Kasey Reardon, Brett Van Dam, Sallyann Vu, Catarina Costa, Veronica Ritchie, Gavin Gagnon, Timothy Bateson, Samantha Rogers, Gregory D. Lyng, Stephen T. Sonis. An assessment of pre-conditioning regimens for optimal hCD34+ hematopoietic stem cell humanization of NSGTMmice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 511.
The intestinal microbiome has become increasingly appreciated as a significant mediator of systemic antitumor immunity/response in both naïve and treatment contexts. In naïve contexts, an intact intestinal microbiome has been demonstrated to enhance tumorigenesis, and its composition to mediate primary tumor growth kinetics. In the context of cancer treatment, antibiotic depletion of the intestinal microbiota has been reported to inhibit the efficacy of cyclophosphamide and that of the immune checkpoint inhibitor αCTLA4. Compositional modulation of the intestinal microbiota has been found to be sufficient to enhance the antitumor efficacy of αPD-L1. Here, we assessed the relative importance of the intestinal microbiota in mediating αPD-L1 antitumor efficacy in a B16.F10.SIY murine model of melanoma, by performing parallel efficacy studies in C57BL/6 germ-free (Taconic) or specific pathogen free (Taconic or Jackson) mice. We observed that αPD-L1 treatment provided significant antitumor efficacy of in Taconic mice carrying an intact microbiome; however, this efficacy was abolished in germ-free Taconic mice. Furthermore, we observed that tumors of Jackson mice carrying an intact but compositionally different microbiome did not respond to αPD-L1 treatment. Phenotyping of local tumor and systemic immune responses, as well as characterization of the intestinal microbiome in responder vs nonresponder animals provided mechanistic insights. Taken together, these observations suggest that rational modulation of the microbiome may enhance response to immune checkpoint inhibition, and indicate that the gastrointestinal microbiome and its composition are critical for the antitumor efficacy of αPD-L1. Citation Format: Benjamin G. Cuiffo, Caitlin S. Parello, Chelsea Ritchie, Katie Pedrick, Alexandra Kury, Catarina Costa, Brett Van Dam, Jonathan Jung, Gregory D. Lyng, Stephen T. Sonis. The gastrointestinal microbiome and its composition are critical for antitumor efficacy of immune checkpoint inhibition by anti-PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2683. doi:10.1158/1538-7445.AM2017-2683
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