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Background aims: Extracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow–derived MSCs, this study focused on improving the MSC-EV production for clinical application. Methods: Independent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. Results: The functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. Conclusions: Standardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.

Supplementary Data

Colorectal cancer is one of the most frequent malignancies worldwide. Despite considerable progress in early detection and treatment, there is still an unmet need for novel antitumor therapies, particularly in advanced colorectal cancer. Regulatory T cells (Treg) are increased in the peripheral blood and tumor tissue of patients with colorectal cancer. Recently, transient ablation of tumor-associated Tregs was shown to foster CD8+ T-cell–mediated antitumoral immunity in murine colorectal cancer models. However, before considering therapies on targeting Tregs in patients with cancer, detailed knowledge of the phenotype and features of tumor-associated Tregs is indispensable. Here, we demonstrate in a murine model of inflammation-induced colorectal cancer that tumor-associated Tregs are mainly of thymic origin and equipped with a specific set of molecules strongly associated with enhanced migratory properties. Particularly, a dense infiltration of Tregs in mouse and human colorectal cancer lesions correlated with increased expression of the orphan chemoattractant receptor GPR15 on these cells. Comprehensive gene expression analysis revealed that tumor-associated GPR15+ Tregs have a Th17-like phenotype, thereby producing IL17 and TNFα. Gpr15 deficiency repressed Treg infiltration in colorectal cancer, which paved the way for enhanced antitumoral CD8+ T-cell immunity and reduced tumorigenesis. In conclusion, GPR15 represents a promising novel target for modifying T-cell–mediated antitumoral immunity in colorectal cancer.Significance:The G protein–coupled receptor 15, an unconventional chemokine receptor, directs Tregs into the colon, thereby modifying the tumor microenvironment and promoting intestinal tumorigenesis.See related commentary by Chakraborty and Zappasodi, p. 2817

PDF file - 149K, Suppl. Figure 1. The induction of colitis-associated colon cancer alters the expression of GARP, CD103 and CTLA-4 on T cells and regulatory T cells. Suppl. Figure 2. Increased percentages of regulatory T cells in the peripheral blood of patients with colon cancer. Suppl. Figure 3. Impact of DT treatment on the distribution of Tregs and adaptive immunity in DEREG mice. Suppl. Figure 4. Impact of DT treatment on the distribution of Tregs in DEREG mice.

PDF file - 204K

Treatment options for pancreatic cancer are limited and often ineffective. Immunotherapeutic approaches are one possible option that needs to be evaluated in appropriate animal models. The aim of the present study was to analyze tumor-specific immune responses in a mouse model of pancreatic cancer, which mimics the human disease closely. C57BL/6 EL-TGF-α × Trp53−/− mice, which develop spontaneous ductal pancreatic carcinoma, were generated. EL-TGF-α × Trp53−/− mice developed spontaneous pancreatic tumors with pathomorphologic features close to the human disease. Tumor-specific CD8+ T-cell responses and IgG responses were analyzed in EL-TGF-α × Trp53−/− mice during tumor development and compared with mice with s.c. growing pancreatic tumors. In contrast to spontaneous pancreatic tumors, cell lines generated from these tumors were rejected after s.c. injection into wild-type mice but not in nude or RAG knockout mice. Direct comparison of spontaneous and s.c. injected tumors revealed an impaired infiltration of CD8+ T cells in spontaneous pancreatic tumors, which was also evident after adoptive transfer of tumor-specific T cells. Intratumoral cytokine secretion of tumor necrosis factor-α, IFN-γ, IL-6, and MCP-1 was lower in spontaneous tumors as well as the number of adoptively transferred tumor-specific T cells. Our data provide clear evidence for tumor-specific immune responses in a genetic mouse model for pancreatic carcinoma. Comparative analysis of s.c. injected tumors and spontaneous tumors showed significant differences in tumor-specific immune responses, which will help in improving current immune-based cancer therapies against adenocarcinoma of the pancreas. (Cancer Res 2006; 66(1): 508-16)

PDF file - 43K, Supplemental Table 1. Characteristics of colon cancer patients included in the study. Supplemental Table 2. Primers.

Supplementary Figure 1 and Table S2 from Genetically Induced Pancreatic Adenocarcinoma Is Highly Immunogenic and Causes Spontaneous Tumor-Specific Immune Responses

Over the past few years, growing evidence suggests direct crosstalk between thyroid hormones (THs) and the immune system. Components of the immune system were proposed to interfere with the central regulation of systemic TH levels. Conversely, THs regulate innate and adaptive immune responses as immune cells are direct target cells of THs. Accordingly, they express different components of local TH action, such as TH transporters or receptors, but our picture of the interplay between THs and the immune system is still incomplete. This review provides a critical overview of current knowledge regarding the interaction of THs and the immune system with the main focus on local TH action within major innate and adaptive immune cell subsets. Thereby, this review aims to highlight open issues which might help to infer the clinical relevance of THs in host defence in the context of different types of diseases such as infection, ischemic organ injury or cancer.

Human cytomegalovirus (HCMV) frequently causes congenital infections, resulting in birth defects and developmental disorders. A vaccine is needed, but unavailable. We analyzed the potential of CMV mutants, lacking their STAT2 antagonists to serve as live attenuated vaccine viruses in mice. Infections with attenuated viruses elicited strong ELISA-reactive binding IgG responses and induced neutralizing antibodies as well as antibodies stimulating cellular Fcγ receptors, including the antibody-dependent cellular cytotoxicity (ADCC)-eliciting receptors FcγRIII/CD16 and FcγRIV. Accordingly, vaccinated mice were fully protected against challenge infections. Female mice vaccinated prior to gestation transmitted CMV-specific IgG to their offspring, which protected the progeny from perinatal infections in a mouse model for congenital CMV disease. To define the role of maternal antibodies, female mice either capable or incapable of producing antibodies were vaccinated and subsequently bred to males of the opposite genotype. Challenge infections of the genotypically identical F1 generation revealed the indispensability of maternal antibodies for vaccine-induced protection against cytomegaloviruses.

A wide range of microbial pathogens is capable of entering the gastrointestinal tract, causing infectious diarrhea and colitis. A finely tuned balance between different cytokines is necessary to eradicate the microbial threat and to avoid infection complications. The current study identified IL-33 as a critical regulator of the immune response to the enteric pathogen Citrobacter rodentium. We observed that deficiency of the IL-33 signaling pathway attenuates bacterial-induced colitis. Conversely, boosting this pathway strongly aggravates the inflammatory response and makes the mice prone to systemic infection. Mechanistically, IL-33 mediates its detrimental effect by enhancing gut permeability and by limiting the induction of protective T helper 17 cells at the site of infection, thus impairing host defense mechanisms against the enteric pathogen. Importantly, IL-33-treated infected mice supplemented with IL-17A are able to resist the otherwise strong systemic spreading of the pathogen. These findings reveal a novel IL-33/IL-17A crosstalk that controls the pathogenesis of Citrobacter rodentium-driven infectious colitis. Manipulating the dynamics of cytokines may offer new therapeutic strategies to treat specific intestinal infections.

Ultrasmall gold nanoparticles (2 nm) easily penetrate the membranes of intestinal murine epithelial cells (MODE-K) and colorectal cancer cells (CT-26). They are also taken up by 3D spheroids (400 mu m) of these cell types and primary gut organoids (500 mu m). In contrast, dissolved dyes are not taken up by any of these cells or 3D structures. The distribution of fluorescent ultrasmall gold nanoparticles inside cells, spheroids, and gut organoids is examined by confocal laser scanning microscopy. Nanoparticles conjugated with the cytostatic drug doxorubicin and a fluorescent dye exhibit significantly greater cytotoxicity toward CT-26 tumor spheroids than equally concentrated dissolved doxorubicin, probably because they enter the interior of a spheroid much more easily than dissolved doxorubicin. Comprehensive analyses show that the cellular uptake of ultrasmall gold nanoparticles occurs by different endocytosis pathways.

CD47 is an ubiquitously expressed surface molecule with significant impact on immune responses. However, its role for antiviral immunity is not fully understood. Here, we revealed that the expression of CD47 on immune cells seemed to disturb the antiviral immune response as CD47-deficient mice (CD47−/−) showed an augmented clearance of influenza A virus (IAV). Specifically, we have shown that enhanced viral clearance is mediated by alveolar macrophages (aMФ). Although aMФ displayed upregulation of CD47 expression during IAV infection in wildtype mice, depletion of aMФ in CD47−/− mice during IAV infection reversed the augmented viral clearance. We have also demonstrated that CD47 restricts hemoglobin (HB) expression in aMФ after IAV and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, with HB showing antiviral properties by enhancing the IFN-β response. Our study showed a negative role for CD47 during antiviral immune responses in the lung by confining HB expression in aMФ.

Clinical and experimental studies indicate that the bacterial and fungal gut microbiota modulates immune responses in distant organs including the lungs. Immune dysregulation is associated with severe SARS-CoV-2 infection, and several groups have observed gut bacterial dysbiosis in SARS-CoV-2 infected patients, while the fungal gut microbiota remains poorly defined in these patients. We analyzed the fungal gut microbiome from rectal swabs taken prior to anti-infective treatment in 30 SARS-CoV-2 positive (21 non-severe COVID-19 and 9 developing severe/critical COVID-19 patients) and 23 SARS-CoV-2 negative patients by ITS2-sequencing. Pronounced but distinct interconnected fungal communities distinguished SARS-CoV-2 positive and negative patients. Fungal gut microbiota in severe/critical COVID-19 illness was characterized by a reduced diversity, richness and evenness and by an increase of the relative abundance of the Ascomycota phylum compared with non-severe COVID-19 illness. A dominance of a single fungal species with a relative abundance of >75% was a frequent feature in severe/critical COVID-19. The dominating fungal species were highly variable between patients even within the groups. Several fungal taxa were depleted in patients with severe/critical COVID-19.The distinct compositional changes of the fungal gut microbiome in SARS-CoV-2 infection, especially in severe COVID-19 illness, illuminate the necessity of a broader approach to investigate whether the differences in the fungal gut microbiome are consequences of SARS-CoV-2 infection or a predisposing factor for critical illness.

The incidence of gastrointestinal infections continues to increase, and infectious colitis contributes significantly to morbidity and mortality worldwide. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) has been discovered to be strongly involved in the intestinal homeostasis. However, whether intestinal CEACAM1 expression has an impact on the control of infectious colitis remains elusive. Citrobacter rodentium (C. rodentium) is a gram-negative enteric pathogen that induces colonic inflammation in mice, with a critical role for CD4(+) T cell but not CD8(+) T cell immunity to primary infection. Here, we show that Ceacam1(−/−) mice are much more susceptible to C. rodentium infection than wildtype mice, which is mediated by a defect in the intestinal barrier and, surprisingly, by a dysregulated CD8(+) T cell but not CD4(+) T cell response in the colon. CEACAM1 expression is essential for the control of CD8(+) T cell immunity, as CEACAM1 deficiency during C. rodentium infection inhibits CD8(+) T cell exhaustion. We conclude that CEACAM1 is an important regulator of CD8(+) T cell function in the colon, and blocking CEACAM1 signaling to activate CD8(+) T cells may have unforeseen side effects.

This paper aims to review data regarding two determinants of comorbidity between depression and obesity, i.e., the role of disturbed gut microbiome in their genesis and of diets in their treatment. Obesity and major depressive disorders (MDD) are highly comorbid, the “metabolic” (obese) subtype of MDD affects about one third of all individuals with MDD. There is an urgent need for better therapies strategies, which may include specific dietary measures. A diet low in carbohydrates (low-carb), effective in obesity, may be beneficial also in MDD. However, the underlying mechanisms have not yet been elucidated. Recent data suggest a key role of gut microbiota, neuroplasticity, and neuroinflammation in obesity and MDD. We will focus on the brain-derived neurotrophic factor (BDNF), and microglial fractalkine, a main modulator of neuroinflammation. BDNF and fractalkine may be involved in “metabolic” depression. Future studies may uncover specific pathophysiological pathways in affected patients towards more efficient causal therapies.

Decline in immune function during aging increases susceptibility to different aging-related diseases. However, the underlying molecular mechanisms, especially the genetic factors contributing to imbalance of naïve/memory T-cell subpopulations, still remain largely elusive. Here, we show that loss of DJ-1 encoded by PARK7/DJ-1, causing early-onset familial Parkinson’s disease (PD), unexpectedly diminished signs of immunoaging in T-cell compartments of both human and mice. Compared with two gender-matched unaffected siblings of similar ages, the index PD patient with DJ-1 deficiency showed a decline in many critical immunoaging features, including almost doubled non-senescent T cells. The observation was further consolidated by the results in 45-week-old DJ-1 knockout mice. Our data demonstrated that DJ-1 regulates several immunoaging features via hematopoietic-intrinsic and naïve-CD8-intrinsic mechanisms. Mechanistically, DJ-1 depletion reduced oxidative phosphorylation (OXPHOS) and impaired TCR sensitivity in naïve CD8 T cells at a young age, accumulatively leading to a reduced aging process in T-cell compartments in older mice. Our finding suggests an unrecognized critical role of DJ-1 in regulating immunoaging, discovering a potent target to interfere with immunoaging- and aging-associated diseases.

Acid sphingomyelinase (Asm) and acid ceramidase (Ac) are parts of the sphingolipid metabolism. Asm hydrolyzes sphingomyelin to ceramide, which is further metabolized to sphingosine by Ac. Ceramide generates ceramide-enriched platforms that are involved in receptor clustering within cellular membranes. However, the impact of cell-intrinsic ceramide on T cell function is not well characterized. By using T cell-specific Asm- or Ac-deficient mice, with reduced or elevated ceramide levels in T cells, we identified ceramide to play a crucial role in T cell function in vitro and in vivo. T cell-specific ablation of Asm in Asmflox/flox/CD4cre mice resulted in enhanced tumor progression associated with impaired T cell responses, whereas Acflox/flox/CD4cre mice showed reduced tumor growth rates and elevated T cell activation compared to the respective controls upon tumor transplantation. Further in vitro analysis revealed that decreased ceramide content supports CD4+ regulatory T cell differentiation and interferes with cytotoxic activity of CD8+ T cells. In contrast, elevated ceramide concentration in CD8+ T cells from Acflox/flox/CD4cre mice was associated with enhanced cytotoxic activity. Strikingly, ceramide co-localized with the T cell receptor (TCR) and CD3 in the membrane of stimulated T cells and phosphorylation of TCR signaling molecules was elevated in Ac-deficient T cells. Hence, our results indicate that modulation of ceramide levels, by interfering with the Asm or Ac activity has an effect on T cell differentiation and function and might therefore represent a novel therapeutic strategy for the treatment of T cell-dependent diseases such as tumorigenesis.

Primary and recurrent cytomegalovirus (CMV) infections frequently cause CMV colitis in immunocompromised as well as inflammatory bowel disease (IBD) patients. Additionally, colitis occasionally occurs upon primary CMV infection in patients who are apparently immunocompetent. In both cases, the underlying pathophysiologic mechanisms are largely elusive - in part due to the lack of adequate access to specimens. We employed the mouse cytomegalovirus (MCMV) model to probe into the association between CMV and colitis. During acute primary MCMV infection of immunocompetent mice, the gut microbial composition was affected within the first 5 days post-infection as manifested by an altered ratio of the Firmicutes to Bacteroidetes phyla. Interestingly, these microbial changes incited with high-titer MCMV replication in the colon, mild crypt necrosis and increased colonic pro-inflammatory cytokine levels. Further analyses revealed that murine and human intestinal epithelial cell line as well as primary intestinal crypt cells/ organoids represent direct targets of CMV accompanied by increased cell mortality upon infection. Accordingly, in vivo MCMV infection disrupted the intestinal epithelial barrier and increased apoptosis of intestinal epithelial cells. In summary, our data show that CMV induces colitis in immunocompetent hosts by altering the intestinal homeostasis.

Invariant NK T (iNKT) cells are implicated in viral clearance; however, their role in hepatitis C virus (HCV) infection remains controversial. Here, iNKT cells were studied during different stages of HCV infection. iNKT cells from patients with acute HCV infection and people who inject drugs (PWID) with chronic or spontaneously resolved HCV infection were characterized by flow cytometry. In a longitudinal analysis during acute HCV infection, frequencies of activated CD38+ iNKT cells reproducibly declined in spontaneously resolving patients, whereas they were persistently elevated in patients progressing to chronic infection. During the first year of infection, the frequency of activated CD38⁺ or CD69+ iNKT cells strongly correlated with alanine transaminase levels with particularly pronounced correlations in spontaneously resolving patients. Increased frequencies of activated iNKT cells in chronic HCV infection were confirmed in cross-sectional analyses of PWID with chronic or spontaneously resolved HCV infection; however, no apparent functional differences were observed with various stimulation protocols. Our data suggest that iNKT cells are activated during acute hepatitis C and that activation is sustained in chronic infection. The correlation between the frequency of activated iNKT cells and alanine transaminase may point toward a role of iNKT cells in liver damage. CA extern

Summary A reciprocal interaction exists between the gut microbiota and the immune system. Regulatory T (Treg) cells are important for controlling immune responses and for maintaining the intestinal homeostasis but their precise influence on the gut microbiota is unclear. We studied the effects of Treg cell depletion on inflammation of the intestinal mucosa and analysed the gut microbiota before and after depletion of Treg cells using the DEpletion of REGulatory T cells (DEREG) mouse model. DNA was extracted from stool samples of DEREG mice and wild‐type littermates at different time‐points before and after diphtheria toxin application to deplete Treg cells in DEREG mice. The V3/V4 region of the 16S rRNA gene was used for studying the gut microbiota with Illumina MiSeq paired ends sequencing. Multidimensional scaling separated the majority of gut microbiota samples from late time‐points after Treg cell depletion in DEREG mice from samples of early time‐points before Treg cell depletion in these mice and from gut microbiota samples of wild‐type mice. Treg cell depletion in DEREG mice was accompanied by an increase in the relative abundance of the phylum Firmicutes and by intestinal inflammation in DEREG mice 20 days after Treg cell depletion, indicating that Treg cells influence the gut microbiota composition. In addition, the variables cage, breeding and experiment number were associated with differences in the gut microbiota composition and these variables should be respected in murine studies., In this study we showed that temporal regulatory T (Treg) cell depletion in DEpletion of REGulatory T cells mice was accompanied by an increase of the relative abundance of the phylum Firmicutes of the gut microbiota, indicating that Treg cells affect the gut microbiota composition. In addition, Treg cell depletion was associated with intestinal inflammation. Furthermore, the variables cage, breeding and experiment number affected the gut microbiota composition and should be respected in murine gut microbiota studies

Therapeutic promotion of intestinal regeneration holds great promise, but defining the cellular mechanisms that influence tissue regeneration remains an unmet challenge. To gain insight into the process of mucosal healing, we longitudinally examined the immune cell composition during intestinal damage and regeneration. B cells were the dominant cell type in the healing colon, and single-cell RNA sequencing (scRNA-seq) revealed expansion of an IFN-induced B cell subset during experimental mucosal healing that predominantly located in damaged areas and associated with colitis severity. B cell depletion accelerated recovery upon injury, decreased epithelial ulceration, and enhanced gene expression programs associated with tissue remodeling. scRNA-seq from the epithelial and stromal compartments combined with spatial transcriptomics and multiplex immunostaining showed that B cells decreased interactions between stromal and epithelial cells during mucosal healing. Activated B cells disrupted the epithelial-stromal cross talk required for organoid survival. Thus, B cell expansion during injury impairs epithelial-stromal cell interactions required for mucosal healing, with implications for the treatment of IBD.

OA Förderung 2021 The gut microbiota contributes to maintaining human health and regulating immune responses. Severe COVID-19 illness is associated with a dysregulated pro-inflammatory immune response. The effect of SARS-CoV-2 on altering the gut microbiome and the relevance of the gut microbiome on COVID-19 severity needs to be clarified. In this prospective study, we analyzed the gut microbiome of 212 patients of a tertiary care hospital (117 patients infected with SARS-CoV-2 and 95 SARS-CoV-2 negative patients) using 16S rRNA gene sequencing of the V3-V4 region. Inflammatory markers and immune cells were quantified from blood. The gut microbiome in SARS-CoV-2 infected patients was characterized by a lower bacterial richness and distinct differences in the gut microbiome composition, including an enrichment of the phyla Proteobacteria and Bacteroidetes and a decrease of Actinobacteria compared to SARS-CoV-2 negative patients. The relative abundance of several genera including Bifidobacterium, Streptococcus and Collinsella was lower in SARS-CoV-2 positive patients while the abundance of Bacteroides and Enterobacteriaceae was increased. Higher pro-inflammatory blood markers and a lower CD8(+) T cell number characterized patients with severe COVID-19 illness. The gut microbiome of patients with severe/critical COVID-19 exhibited a lower abundance of butyrate-producing genera Faecalibacterium and Roseburia and a reduction in the connectivity of a distinct network of anti-inflammatory genera that was observed in patients with mild COVID-19 illness and in SARS-CoV-2 negative patients. Dysbiosis of the gut microbiome associated with a pro-inflammatory signature may contribute to the hyperinflammatory immune response characterizing severe COVID-19 illness.

OA Förderung 2021 The hallmarks of inflammatory bowel disease are mucosal damage and ulceration, which are known to be high-risk conditions for the development of colorectal cancer. Recently, interleukin (IL)-33 and its receptor ST2 have emerged as critical modulators in inflammatory disorders. Even though several studies highlight the IL-33/ST2 pathway as a key factor in colitis, a detailed mode of action remains elusive. Therefore, we investigated the role of IL-33 during intestinal inflammation and its potential as a novel therapeutic target in colitis. Interestingly, the expression of IL-33, but not its receptor ST2, was significantly increased in biopsies from the inflamed colon of IBD patients compared to non-inflamed colonic tissue. Accordingly, in a mouse model of Dextran Sulfate Sodium (DSS) induced colitis, the secretion of IL-33 significantly accelerated in the colon. Induction of DSS colitis in ST2-/- mice displayed an aggravated colon pathology, which suggested a favorable role of the IL 33/ST2 pathway during colitis. Indeed, injecting rmIL-33 into mice suffering from acute DSS colitis, strongly abrogated epithelial damage, pro-inflammatory cytokine secretion, and loss of barrier integrity, while it induced a strong increase of Th2 associated cytokines (IL-13/IL-5) in the colon. This effect was accompanied by the accumulation of regulatory T cells (Tregs) and type 2 innate lymphoid cells (ILC2s) in the colon. Depletion of Foxp3+ Tregs during IL-33 treatment in DSS colitis ameliorated the positive effect on the intestinal pathology. Finally, IL-33 expanded ILC2s, which were adoptively transferred to DSS treated mice, significantly reduced colonic inflammation compared to DSS control mice. In summary, our results emphasize that the IL-33/ST2 pathway plays a crucial protective role in colitis by modulating ILC2 and Treg numbers.

Patients suffering from ulcerative colitis are at increased risk of developing colorectal cancer. Although the exact underlying mechanisms of inflammation-associated carcinogenesis remain unknown, the intestinal microbiota as well as pathogenic bacteria are discussed as contributors to inflammation and colitis-associated colon cancer (CAC). In the present study, we analyzed the impact of TLR4, the receptor for Gram-negative bacteria derived lipopolysaccharides, on intestinal inflammation and tumorigenesis in a murine model of CAC. During the inflammatory phases of CAC development, we observed a strong upregulation of Tlr4 expression in colonic tissues. Blocking of TLR4 signaling by a small-molecule-specific inhibitor during the inflammatory phases of CAC strongly diminished the development and progression of colonic tumors, which was accompanied by decreased numbers of infiltrating macrophages and reduced colonic pro-inflammatory cytokine levels compared to CAC control mice. Interestingly, inhibiting bacterial signaling by antibiotic treatment during the inflammatory phases of CAC also protected mice from severe intestinal inflammation and almost completely prevented tumor growth. Nevertheless, application of antibiotics involved rapid and severe body weight loss and might have unwanted side effects. Our results indicate that bacterial activation of TLR4 on innate immune cells in the colon triggers inflammation and promotes tumor growth. Thus, the inhibition of the TLR4 signaling during intestinal inflammation might be a novel approach to impede CAC development. OA Förderung 2021

Neuropilin-1 (Nrp-1) is a well described marker molecule for CD4

Despite significant efforts, vaccines are not yet available for every infectious pathogen, and the search for a protective approach to prevent the establishment of chronic infections, i.e., with HIV, continues. Immune checkpoint therapies targeting inhibitory receptors, such as PD-1, have shown impressive results against solid tumors., PD-1-targeted therapies have shown modest antiviral effects in preclinical models of chronic viral infection. Thus, novel therapy protocols are necessary to enhance T cell immunity and viral control to overcome T cell dysfunction and immunosuppression. Here, we demonstrate that nanoparticle-based therapeutic vaccination improved PD-1-targeted therapy during chronic infection with Friend retrovirus (FV). Prevention of inhibitory signals by blocking PD-L1 in combination with therapeutic vaccination with nanoparticles containing the microbial compound CpG and a CD8+ T cell Gag epitope peptide synergistically enhanced functional virus-specific CD8+ T cell responses and improved viral clearance. We characterized the CD8+ T cell populations that were affected by this combination therapy, demonstrating that new effector cells were generated and that exhausted CD8+ T cells were reactivated at the same time. While CD8+ T cells with high PD-1 (PD-1hi) expression turned into a large population of granzyme B-expressing CD8+ T cells after combination therapy, CXCR5-expressing follicular cytotoxic CD8+ T cells also expanded to a high degree. Thus, our study describes a very efficient approach to enhance virus control and may help us to understand the mechanisms of combination immunotherapy reactivating CD8+ T cell immunity. A better understanding of CD8+ T cell immunity during combination therapy will be important for developing efficient checkpoint therapies against chronic viral infections and cancer.

Epigenetic modifications play critical roles in regulating cell lineage differentiation, but the epigenetic mechanisms guiding specific differentiation steps within a cell lineage have rarely been investigated. To decipher such mechanisms, we used the defined transition from proliferating (PC) into hypertrophic chondrocytes (HC) during endochondral ossification as a model. We established a map of activating and repressive histone modifications for each cell type. ChromHMM state transition analysis and Pareto-based integration of differential levels of mRNA and epigenetic marks revealed that differentiation-associated gene repression is initiated by the addition of H3K27me3 to promoters still carrying substantial levels of activating marks. Moreover, the integrative analysis identified genes specifically expressed in cells undergoing the transition into hypertrophy. Investigation of enhancer profiles detected surprising differences in enhancer number, location, and transcription factor binding sites between the two closely related cell types. Furthermore, cell type-specific upregulation of gene expression was associated with increased numbers of H3K27ac peaks. Pathway analysis identified PC-specific enhancers associated with chondrogenic genes, whereas HC-specific enhancers mainly control metabolic pathways linking epigenetic signature to biological functions. Since HC-specific enhancers show a higher conservation in postnatal tissues, the switch to metabolic pathways seems to be a hallmark of differentiated tissues. Surprisingly, the analysis of H3K27ac levels at super-enhancers revealed a rapid adaption of H3K27ac occupancy to changes in gene expression, supporting the importance of enhancer modulation for acute alterations in gene expression. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Colorectal cancer is one of the most frequent malignancies worldwide. Despite considerable progress in early detection and treatment, there is still an unmet need for novel antitumor therapies, particularly in advanced colorectal cancer. Regulatory T cells (Treg) are increased in the peripheral blood and tumor tissue of patients with colorectal cancer. Recently, transient ablation of tumor-associated Tregs was shown to foster CD8+ T-cell–mediated antitumoral immunity in murine colorectal cancer models. However, before considering therapies on targeting Tregs in patients with cancer, detailed knowledge of the phenotype and features of tumor-associated Tregs is indispensable. Here, we demonstrate in a murine model of inflammation-induced colorectal cancer that tumor-associated Tregs are mainly of thymic origin and equipped with a specific set of molecules strongly associated with enhanced migratory properties. Particularly, a dense infiltration of Tregs in mouse and human colorectal cancer lesions correlated with increased expression of the orphan chemoattractant receptor GPR15 on these cells. Comprehensive gene expression analysis revealed that tumor-associated GPR15+ Tregs have a Th17-like phenotype, thereby producing IL17 and TNFα. Gpr15 deficiency repressed Treg infiltration in colorectal cancer, which paved the way for enhanced antitumoral CD8+ T-cell immunity and reduced tumorigenesis. In conclusion, GPR15 represents a promising novel target for modifying T-cell–mediated antitumoral immunity in colorectal cancer. Significance: The G protein–coupled receptor 15, an unconventional chemokine receptor, directs Tregs into the colon, thereby modifying the tumor microenvironment and promoting intestinal tumorigenesis. See related commentary by Chakraborty and Zappasodi, p. 2817

Neuropilin-1 (Nrp-1) is a well described marker molecule for CD4+Foxp3+ thymus-derived regulatory T cells (Tregs). In addition, a small population of CD4+Foxp3− conventional (conv) T cells expresses Nrp-1 in naive mice, and Nrp-1 expression has been described to be upregulated on activated CD4+ T cells. However, the function of Nrp-1 expression on CD4+ non-Tregs still remains elusive. In this study, we demonstrate that Nrp-1 expression was induced upon stimulation of CD4+Foxp3− T cells in vitro and during an ongoing immune response in vivo. This activation-induced Nrp-1+CD4+ T cell subset (iNrp-1+) showed a highly activated phenotype in terms of elevated CD25 and CD44 expression, enhanced production of proinflammatory cytokines, and increased proliferation compared with the Nrp-1−CD4+ counterpart. In contrast, Nrp-1+CD4+Foxp3− conv T cells from naive mice (nNrp-1+) were dysfunctional. nNrp-1+CD4+ conv T cells upregulated activation-associated molecules to a lesser extent, exhibited impaired proliferation and produced fewer proinflammatory cytokines than Nrp-1−CD4+ conv T cells upon stimulation in vitro. Moreover, the expression of PD-1 and CTLA-4 was significantly higher on nNrp-1+CD4+Foxp3− T cells compared with iNrp-1+CD4+Foxp3− T cells and Nrp-1−CD4+Foxp3− T cells after stimulation and under homeostatic conditions. Strikingly, transfer of Ag-specific iNrp-1+CD4+ conv T cells aggravated diabetes development, whereas Ag-specific nNrp-1+CD4+ conv T cells failed to induce disease in a T cell transfer model of diabetes. Overall, our results indicate that Nrp-1 expression has opposite functions in recently activated CD4+ non-Tregs compared with CD4+ non-Tregs from naive mice.

Extracellular vesicles (EVs) harvested from cell culture supernatants of human mesenchymal stromal cells (MSCs) suppress acute inflammation in preclinical models of various diseases. Furthermore, they promote regeneration of damaged tissues. Following successful clinical treatment of a steroid-refractory Graft-versus-Host-Disease (GvHD) patient with EVs prepared from conditioned media of human bone marrow (BM)-derived MSCs, we aim to improve MSC-EV production and quality control towards clinical application. Observing functional differences of independent MSC-EV preparations in vitro, we established an optimized murine GvHD model for the analysis of independent MSC-EV preparations in vivo. To this end, T cell depleted allogeneic BM cells co-transplanted with naive allogeneic spleen-derived T cells induced GvHD symptoms with reproducible strengths in mice being preconditioned by ionizing irradiation. Administration of MSC-EV preparations with confirmed in vitro immune modulatory properties at three consecutive days significantly suppressed GvHD symptoms. In contrast, application of MSC-EV preparations lacking these in vitro immune modulating capabilities failed to suppress GvHD symptoms. Thus, our results reveal therapeutic differences among independent MSC-EV preparations that had been produced in a standardized manner. Thus, given this functional heterogeneity, any individual MSC-EV preparation considered for the clinical application should be evaluated for its potency prior to administration to patients.

Heroin dependence may result in suppression of adaptive immune responses. Previously, we demonstrated an increase in relative numbers of inhibitory CD4⁺ regulatory T cells (Tregs) and impaired proliferative activity of CD4⁺ T cells from heroin-addicted patients in contrast to patients in opioid maintenance therapy and healthy controls. However, it remains elusive whether heroin has a direct impact on the CD4⁺ T cell compartment or whether observed effects result from stressful living conditions. Here, we analyzed the frequencies of Tregs and the proliferation as well as the cytokine production of stimulated CD4⁺ T cells from heroin-addicted patients with use of illicit heroin, patients in heroin-assisted treatment (HAT), and patients in methadone maintenance therapy (MMT). Relative numbers of CD4⁺ Tregs were significantly enhanced in patients with illicit heroin abuse compared with patients in HAT or MMT. Notably, CD4⁺ T cells from patients in HAT and from persons using illicit heroin showed significant reduced proliferation and secretion of the pro-inflammatory cytokines IFN-γ and IL-6 upon stimulation in vitro. From these results, we conclude that structured programs such as HAT and MMT dampen elevated frequencies of Tregs in heroin-addicted patients, whereas chronic heroin administration irrespective of using illicit heroin or receiving HAT has a direct impact on the proliferative activity and cytokine production of CD4⁺ T cells.

Heroin dependence may result in suppression of adaptive immune responses. Previously, we demonstrated an increase in relative numbers of inhibitory CD4