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Conventional cancer chemotherapies are not fully efficacious and do not target tumors, leading to significant treatment-related morbidities. A number of genetically attenuated cancer-targeting bacteria are being developed to safely target tumors in vivo. Here we report the toxicological, tumor-targeting, and efficacy profiles of Salmonella enterica serovar Typhimurium CRC2631 in a syngeneic and autochthonous TRAMP model of aggressive prostate cancer. CRC2631 preferentially colonize primary and metastatic tumors in the TRAMP animals. In addition, longitudinal whole genome sequencing studies of CRC2631 recovered from prostate tumor tissues demonstrate that CRC2631 is genetically stable. Moreover, tumor-targeted CRC2631 generates an anti-tumor immune response. Combination of CRC2631 with checkpoint blockade reduces metastasis burden. Collectively, these findings demonstrate a potential for CRC2631 in cancer immunotherapy strategies.

Background. Heat shock proteins (Hsp) act as intracellular chaperones and in addition are used as adjuvant in vaccines of peptides complexed with recombinant Hsp. By interacting with autologous peptides, Hsp may promote the induction of autoimmune reactivity. Objective. Here, we analysed whether the effect of Hsp on macrophages is modulated by insulin peptides known to interact with Hsp. Results. Combinations of the 70 kDa Hsp DnaK with peptide B11-23 from the core region of the proinsulin B-chain induced the release of the inflammatory mediators interleukin-6, tumor necrosis factor α, and interleukin-1β from cells of human and murine macrophage lines. In parallel, there was high-affinity binding of B11-23 to DnaK. DnaK mixed with peptides from other regions of the insulin molecule did not stimulate cytokine secretion. DnaK alone induced little cytokine production, and peptides alone induced none. Conclusion. The macrophage-stimulating potential of Hsp70 family proteins when combined with the proinsulin B-chain peptide B11-23 may contribute to the immunodominance of this peptide in the development of beta cell-directed autoimmunity in type 1 diabetes.

The incidence of type 1 diabetes (T1D) is determined by both genetic and environmental factors. In recent years, the gut microbiota have been identified to be an important environmental factor that could modify diabetes susceptibility. We have previously shown that Myeloid differentiation primary response gene 88 (MyD88), a major adaptor protein downstream of most innate immune Toll-like receptor (TLR) signaling, is important for mediating diabetes susceptibility in the non-obese diabetic (NOD) mouse model of human T1D. Here we report the role of TIR-domain-containing adapter-inducing interferon-β (TRIF) in T1D development, as TRIF is an important adaptor protein downstream of TLR3 and TLR4 signaling. We found that TRIF-deficient (TRIF(−/−)) NOD mice were protected from development of diabetes, but only when housed with TRIF-deficient (TRIF(−/−)) NOD mice. When housed with TRIF-sufficient wild type (WT, i.e., TRIF(+/+)) NOD mice, the mice developed diabetes. We further investigated the gut microbiota as a potential cause for the altered diabetes development. Interestingly, TRIF(−/−)NOD mice had a different microbiota composition compared to WT NOD mice, only if they were housed with TRIF(−/−)NOD mice. However, the composition of gut microbiota in the TRIF(−/−)NOD mice was indistinguishable from WT NOD mice, if they were housed with WT NOD mice. The difference in the gut microbiota in TRIF(−/−)NOD mice, due to cohousing, accorded with the diabetes development in TRIF(−/−)NOD mice. Comparing the gut microbiota in TRIF(−/−) and WT NOD mice, we identified changes in percentage of Sutterella, Rikenella and Turicibacter species. Moreover, bacteria from WT NOD mice induced significantly stronger inflammatory immune responses in vitro compared to those from TRIF(−/−)NOD mice. Further immunological analysis revealed impaired function of dendritic cells and reduced T cell activation and proliferation in TRIF(−/−)NOD mice. Our data show that TRIF-deficiency protects NOD mice from diabetes development through alteration of the gut microbiota and reduced immune cell activation; however, that protection is over-ridden upon exposure to WT NOD bacteria. Therefore exposure to different microbiota can modify disease susceptibility determined by genetic factors related to innate immunity.

Summary Autoimmune hepatitis (AIH) is a chronic liver disease characterized by progressive inflammation, female preponderance and seropositivity for autoantibodies such as anti-smooth muscle actin and/or anti-nuclear, anti-liver kidney microsomal type 1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1) in more than 80% of cases. AIH is linked strongly to several major histocompatibility complex (MHC) alleles, including human leucocyte antigen (HLA)-DR3, -DR7 and -DR13. HLA-DR4 has the second strongest association with adult AIH, after HLA-DR3. We investigated the role of HLA-DR4 in the development of AIH by immunization of HLA-DR4 (DR4) transgenic non-obese diabetic (NOD) mice with DNA coding for human CYP2D6/FTCD fusion autoantigen. Immunization of DR4 mice leads to sustained mild liver injury, as assessed biochemically by elevated alanine aminotransferase, histologically by interface hepatitis, plasma cell infiltration and mild fibrosis and immunologically by the development of anti-LKM1/anti-LC1 antibodies. In addition, livers from DR4 mice had fewer regulatory T cells (Tregs), which had decreased programmed death (PD)-1 expression. Splenic Tregs from these mice also showed impaired inhibitory capacity. Furthermore, DR4 expression enhanced the activation status of CD8+ T cells, macrophages and dendritic cells in naive DR4 mice compared to naive wild-type (WT) NOD mice. Our results demonstrate that HLA-DR4 is a susceptibility factor for the development of AIH. Impaired suppressive function of Tregs and reduced PD-1 expression may result in spontaneous activation of key immune cell subsets, such as antigen-presenting cells and CD8+ T effectors, facilitating the induction of AIH and persistent liver damage.

The incidence of type 1 diabetes (T1D) is rising drastically for the past decades at a rate that cannot be explained by genetic changes alone. Environmental changes are considered to be the main drivers of this change. Recently, the gut microbiota has been suggested as a missing link between known environmental disease modulators and T1D promotion. Lifestyle factors have changed over time and have altered the gut microbiota-host interaction affecting T1D development. The purpose of this review is to discuss recent data emphasizing the modulatory potential of early lifestyle factors on gut microbiota and to elucidate their implication for T1D. Recent findings show that lifestyle factors, especially those that affect the early establishment of gut homeostasis and the education of the immune system, are crucial disease modulators. Changing lifestyle factors affecting the early establishment of gut homeostasis are suggested to be key drivers of the rising T1D incidence.

Type 1 Diabetes (T1D) is a multifactorial, immune-mediated disease, which is characterized by the progressive destruction of autologous insulin-producing beta cells in the pancreas. The risk of developing T1D is determined by genetic, epigenetic and environmental factors. In the past few decades there has been a continuous rise in the incidence of T1D, which cannot be explained by genetic factors alone. Changes in our lifestyle that include diet, hygiene, and antibiotic usage have already been suggested to be causal factors for this rising T1D incidence. Only recently have microbiota, which are affected by all these factors, been recognized as key environmental factors affecting T1D development. In this review we will summarize current knowledge on the impact of gut microbiota on T1D development and give an outlook on the potential to design new microbiota-based therapies in the prevention and treatment of T1D.

The microbiome affects development and activity of the immune system, and may modulate immune therapies, but there is little direct information about this control in vivo. We studied how the microbiome affects regulation of human immune cells in humanized mice. When humanized mice were treated with a cocktail of 4 antibiotics, there was an increase in the frequency of effector T cells in the gut wall, circulating levels of IFN-γ, and appearance of anti-nuclear antibodies. Teplizumab, a non–FcR-binding anti-CD3ε antibody, no longer delayed xenograft rejection. An increase in CD8+ central memory cells and IL-10, markers of efficacy of teplizumab, were not induced. IL-10 levels were only decreased when the mice were treated with all 4 but not individual antibiotics. Antibiotic treatment affected CD11b+CD11c+ cells, which produced less IL-10 and IL-27, and showed increased expression of CD86 and activation of T cells when cocultured with T cells and teplizumab. Soluble products in the pellets appeared to be responsible for the reduced IL-27 expression in DCs. Similar changes in IL-10 induction were seen when human peripheral blood mononuclear cells were cultured with human stool samples. We conclude that changes in the microbiome may impact the efficacy of immunosuppressive medications by altering immune regulatory pathways.

Wen et al. show that there are gut microbial antigens sharing significant homology with the host pancreatic protein peptide IGRP and that can drive self-reactive T cell activation and accelerate diabetes in NOD mice., Both animal model and human studies indicate that commensal bacteria may modify type 1 diabetes (T1D) development. However, the underlying mechanisms by which gut microbes could trigger or protect from diabetes are not fully understood, especially the interaction of commensal bacteria with pathogenic CD8 T cells. In this study, using islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP)–reactive CD8 T cell receptor NY8.3 transgenic nonobese diabetic mice, we demonstrated that MyD88 strongly modulates CD8+ T cell–mediated T1D development via the gut microbiota. Some microbial protein peptides share significant homology with IGRP. Both the microbial peptide mimic of Fusobacteria and the bacteria directly activate IGRP-specific NY8.3 T cells and promote diabetes development. Thus, we provide evidence of molecular mimicry between microbial antigens and an islet autoantigen and a novel mechanism by which the diabetogenicity of CD8+ T cells can be regulated by innate immunity and the gut microbiota.

Adipocytes represent an important cellular source of inflammatory mediators. However, the signals for the induction of proinflammatory adipocyte activities are largely unknown. Here, we demonstrate that heat shock protein (Hsp) 60, a potent stimulator of innate immunity, induces the release of the inflammatory mediators interleukin-6, CXCL1 and monocyte chemoattractant protein-1 in a time- and concentration-dependent manner from cells of the adipocyte line 3T3-L1 and from adipocytes of obese mice. These results identify Hsp60 as an important regulator of adipocyte functions which contribute to the development of inflammatory processes as observed in diabetes and diabetes-associated complications.

Toll-like receptors: Key receptors of the innate immune system The human body constantly encounters a diverse spectrum of pathogens. To defend itself, a complex immune system has evolved consisting of two subdivisions: the innate and the adaptive immune systems. The innate immune system constitutes the so-called “first line of defense” and acts through highly conserved germ-line-encoded pattern recognition receptors (PRRs) (1). These receptors bind to common pathogen-associated molecular patterns (PAMPs), which are vital for the survival of the microorganisms and cannot be altered by mutations. Pattern recognition receptors were first discovered in the fruit fly Drosophila melanogaster. The PRRs in Drosophila, named Toll, play an important role in the recognition of microorganisms such as fungi as well as coordinating embryonic development of the dorso-ventral axis. Homologs of Toll in vertebrates are called Toll-like receptors (TLRs). The first TLR in humans was described in 1997 (2). To date, another 9 TLRs have been identified in humans and 13 in mice. Among these, only murine TLR10 is non-functional due to retrovirus insertion (3). Except for TLRs 3, 7, 8, and 9, which are expressed intracellularly, TLRs are located on the surface of innate and adaptive immune cells as well as on non-immune cells such as muscle cells, epithelial cells, adipocytes, and pancreatic beta cells (1). Cell-surface TLRs detect exogenous lipids, lipoproteins, and proteins from microbes; intracellular TLRs recognize bacterial and viral nucleic acids (3). Recognition of their cognate ligand triggers a complex signaling cascade that ultimately results in the induction of various pro-inflammatory chemokines and cytokines and the activation of the adaptive immune system. However, the activation of TLRs and the subsequent induction of inflammatory immune responses are not always beneficial to the host. TLRs are involved in the pathogenesis of various autoimmune and non-infective inflammatory diseases such as systemic lupus erythematosus, multiple sclerosis, arteriosclerosis, inflammatory bowel disease, diabetes, allergy, and cancer (4, 5). Their activation can either attenuate or boost the course of disease by inducing tolerance or triggering autoreactivity, respectively. The role of the TLRs in the pathogenesis of autoimmune-mediated diabetes, also referred to as type 1 diabetes (T1D), has been extensively studied (Table ​(Table1).1). T1D is a T-cell-mediated metabolic disorder with progressive destruction of insulin-producing pancreatic β cells (6). During the course of disease development, diabetogenic T-cells, macrophages, and dendritic cells will infiltrate the pancreatic islets and cause islet inflammation and eventually β cell loss. Table 1 TLR-related studies in the field of T1D. This opinion letter focuses on the beneficial and detrimental aspects of TLR induction in the course of T1D development.

Background/objectiveToll-like receptors (TLR) mediate the recognition of microbial constituents and stress-induced endogenous ligands by the immune system. They may also be involved in the maintenance or break down of tolerance against autologous antigens. The aim of our investigation was to study the consequence of TLR4 deficiency on the development of insulin-deficient diabetes in the NOD mouse.MethodsThe TLR4 defect of the C57BL/10ScN mouse was backcrossed onto the NOD background and the effect of TLR4 deficiency on diabetes development was analysed by in vivo and in vitro studies.ResultsCompared to animals with wildtype TLR4 expression (TLR4(+/+)), female NOD mice carrying a homozygous TLR4 defect (TLR4(-/-)), showed significant acceleration of diabetes development, with a younger age at diabetes onset (TLR4 (+/+) 177±22 d, TLR(-/-): 118±21 d; pConclusionsOur findings demonstrate that TLR4 deficiency results in an acceleration of diabetes development and immune cell infiltration of islets in NOD mice. We conclude that TLR4 is involved in the progression of the insulitis process thereby controlling the development of insulin-deficient diabetes in NOD mice.

Adipocytes play important roles in lipid metabolism but also in the control of inflammatory processes. Based on our previous findings of heat shock protein (Hsp) 60-induced activation of preadipocytes we investigated whether the capacity of heat shock protein 60 (Hsp60) to interact with adipocytes and to stimulate their proinflammatory activity is determined by the differentiation state of the cells. Hsp60 bound to adipocytes and stimulated the release of inflammatory mediators independent of their differentiation state. Hsp60-adipocyte interactions revealed basic characteristics of a receptor-mediated process. Our findings characterize Hsp60 binding and Hsp60-induced release of proinflammatory mediators as fundamental properties of adipocytes independent of their differentiation state.