Mycobacterium vaccae protects against glucocorticoid resistance resulting from combined physical and psychosocial trauma in mice.

Stress-related somatic and psychiatric disorders are often associated with a decline in regulatory T cell (Treg) counts and chronic low-grade inflammation. Recent preclinical evidence suggests that the latter is at least partly mediated by stress-induced upregulation of toll-like receptor (TLR)2 in newly generated neutrophils and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), as well as glucocorticoid (GC) resistance in predominantly PMN-MDSCs following stress-induced upregulation of TLR4 expression. Here we show in mice exposed to the chronic subordinate colony housing (CSC) paradigm that repeated intragastric (i.g.) administrations of a heat-killed preparation of Mycobacterium vaccae NCTC 11659, a saprophytic microorganism with immunoregulatory properties, protected against the stress-induced reduction in systemic Tregs, increase in basal and LPS-induced in vitro splenocyte viability, as well as splenic in vitro GC resistance. Our findings further support the hypothesis that i.g. M. vaccae protects against CSC-associated splenic GC resistance via directly affecting the myeloid compartment, thereby preventing the CSC-induced upregulation of TLR4 in newly generated PMN-MDSCs. In contrast, the protective effects of i.g. M. vaccae on the CSC-induced upregulation of TLR2 in neutrophils and the subsequent increase in basal and LPS-induced in vitro splenocyte viability seems to be indirectly mediated via the Treg compartment. These data highlight the potential for use of oral administration of M. vaccae NCTC 11659 to prevent stress-induced exaggeration of inflammation, a risk factor for development of stress-related psychiatric disorders.

The PMN-MDSC - A key player in glucocorticoid resistance following combined physical and psychosocial trauma.

Stress-associated somatic and psychiatric disorders are often linked to non-resolving low-grade inflammation, which is promoted at least in part by glucocorticoid (GC) resistance of distinct immune cell subpopulations. While the monocyte/macrophage compartment was in the focus of many clinical and preclinical studies, the role of myeloid-derived suppressor cells (MDSCs) in stress-associated pathologies and GC resistance is less understood. As GC resistance is a clear risk factor for posttraumatic complications in patients on intensive care, the exact interplay of physical and psychosocial traumatization in the development of GC resistance needs to be further clarified. In the current study we employ the chronic subordinate colony housing (CSC) paradigm, a well-characterized mouse model of chronic psychosocial stress, to study the role of myeloid cells, in particular of MDSCs, in innate immune activation and GC resistance following combined psychosocial and physical (e.g., bite wounds) trauma. Our findings support the hypothesis that stress-induced neutrophils, polymorphonuclear (PMN)-MDSCs and monocytes/monocyte-like (MO)-MDSCs get primed and activated locally in the bone marrow as determined by toll-like receptor (TLR)2 upregulation and increased basal and lipopolysaccharide (LPS)-induced in vitro cell viability. These primed and activated myeloid cells emigrate into the peripheral circulation and subsequently, if CSC is accompanied by significant bite wounding, accumulate in the spleen. Here, PMN-MDSCs and monocytes/MO-MDSCs upregulate TLR4 expression, which exclusively in PMN-MDSCs promotes NF-κB hyperactivation upon LPS-stimulation, thereby exceeding the anti-inflammatory capacities of GCs and resulting in GC resistance.

TLR4-dependent shaping of the wound site by MSCs accelerates wound healing.

We here address the question whether the unique capacity of mesenchymal stem cells to re-establish tissue homeostasis depends on their potential to sense pathogen-associated molecular pattern and, in consequence, mount an adaptive response in the interest of tissue repair. After injection of MSCs primed with the bacterial wall component LPS into murine wounds, an unexpected acceleration of healing occurs, clearly exceeding that of non-primed MSCs. This correlates with a fundamental reprogramming of the transcriptome in LPS-treated MSCs as deduced from RNAseq analysis and its validation. A network of genes mediating the adaptive response through the Toll-like receptor 4 (TLR4) pathway responsible for neutrophil and macrophage recruitment and their activation profoundly contributes to enhanced wound healing. In fact, injection of LPS-primed MSCs silenced for TLR4 fails to accelerate wound healing. These unprecedented findings hold substantial promise to refine current MSC-based therapies for difficult-to-treat wounds.

miR-146a regulates insulin sensitivity via NPR3.

The pathogenesis of obesity-related metabolic diseases has been linked to the inflammation of white adipose tissue (WAT), but the molecular interconnections are still not fully understood. MiR-146a controls inflammatory processes by suppressing pro-inflammatory signaling pathways. The aim of this study was to characterize the role of miR-146a in obesity and insulin resistance. MiR-146a-/- mice were subjected to a high-fat diet followed by metabolic tests and WAT transcriptomics. Gain- and loss-of-function studies were performed using human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. Compared to controls, miR-146a-/- mice gained significantly more body weight on a high-fat diet with increased fat mass and adipocyte hypertrophy. This was accompanied by exacerbated liver steatosis, insulin resistance, and glucose intolerance. Likewise, adipocytes transfected with an inhibitor of miR-146a displayed a decrease in insulin-stimulated glucose uptake, while transfecting miR-146a mimics caused the opposite effect. Natriuretic peptide receptor 3 (NPR3) was identified as a direct target gene of miR-146a in adipocytes and CRISPR/Cas9-mediated knockout of NPR3 increased insulin-stimulated glucose uptake and enhanced de novo lipogenesis. In summary, miR-146a regulates systemic and adipocyte insulin sensitivity via downregulation of NPR3.

Distinct Effects of IL-6 Classic and Trans-Signaling in Bone Fracture Healing.

Bone healing is a complex process with closely linked phases of inflammation, regeneration, and remodeling. IL-6 may crucially regulate this process; however, the underlying mechanisms are unclear. IL-6 signals are transmitted via the transmembrane glycoprotein 130 by two distinct mechanisms: classic signaling using the membrane-anchored IL-6 receptor and trans-signaling using its soluble form. Herein, we investigated the hypothesis that IL-6 classic and trans-signaling have different functions during bone healing. To investigate fracture healing, 12-week-old C57BL/6J mice underwent a femur osteotomy. To study the function of IL-6 during the inflammatory phase, either an anti-IL-6 antibody, which inhibits IL-6 classic and trans-signaling, or soluble glycoprotein 130 fusion protein, which selectively blocks trans-signaling, was injected after 30 minutes and 48 hours. To analyze IL-6 effects in the repair phase, compounds were injected from day 7 onwards. Global IL-6 inhibition in the early phase after fracture reduced systemic inflammation, the recruitment of immune cells, and bone regeneration, resulting in delayed fracture healing. Global IL-6 inhibition during the repair phase disturbed bone formation and remodeling. In contrast, inhibition of IL-6 trans-signaling exerted minor effects on the immune response and did not influence bone repair, suggesting that the classic pathway accounts for most of the effects observed after global IL-6 inhibition. Our results reveal that IL-6 classic signaling, but not IL-6 trans-signaling, is essential for bone repair.

In vitro-generated MDSCs prevent murine GVHD by inducing type 2 T cells without disabling antitumor cytotoxicity.

Myeloid-derived suppressor cells (MDSCs) inhibit T-cell expansion and functions by versatile mechanisms such as nutrient depletion, nitrosylation, or apoptosis. Since graft-versus-host disease (GVHD) is characterized by the expansion of donor-derived T cells destroying recipient tissue, we analyzed whether MDSCs can be used for GVHD prevention in murine allogeneic bone marrow transplantation models. Transplantation of MDSCs, generated from bone marrow cells by granulocyte-macrophage colony-stimulating factor (GM-CSF)/G-CSF in vitro, inhibited GVHD-induced death and attenuated histologic GVHD, whereas antitumor cytotoxicity of alloantigen-specific T cells was maintained. MDSCs expanded in vivo and invaded lymphatic and GVHD target organs. Major histocompatibility complex class I expression on MDSCs was dispensable for their suppressive capacity. Inhibition of GVHD required the presence of MDSCs during T-cell priming, whereas allogeneic T-cell numbers and homing in lymphoid and GVHD target organs were not considerably affected in MDSC-treated mice. However, MDSCs skewed allogeneic T cells toward type 2 T cells upregulating T helper 2 (Th2)-specific cytokines. Type 2 T-cell induction was indispensable for GVHD prevention since MDSC treatment failed to prevent GVHD when allogeneic STAT6-deficient T cells, which are unable to differentiate into Th2 cells, were transplanted. MDSC-induced Th2 induction might be applicable for GVHD treatment in clinical settings.

A paired comparison between glioblastoma "stem cells" and differentiated cells.

Cancer stem cells (CSC) have been postulated to be responsible for the key features of a malignancy and its maintenances, as well as therapy resistance, while differentiated cells are believed to make up the rapidly growing tumour bulk. It is therefore important to understand the characteristics of those two distinct cell populations in order to devise treatment strategies which effectively target both cohorts, in particular with respect to cancers, such as glioblastoma. Glioblastoma is the most common primary brain tumour in adults, with a mean patient survival of 12-15 months. Importantly, therapeutic improvements have not been forthcoming in the last decade. In this study we compare key features of three pairs of glioblastoma cell populations, each pair consisting of stem cell-like and differentiated cells derived from an individual patient. Our data suggest that while growth rates and expression of key survival- and apoptosis-mediating proteins are more similar according to differentiation status than genetic similarity, we found no intrinsic differences in response to standard therapeutic interventions, namely exposure to radiation or the alkylating agent temozolomide. Interestingly, we could demonstrate that both stem cell-like and differentiated cells possess the ability to form stem cell-containing tumours in immunocompromised mice and that differentiated cells could potentially be dedifferentiated to potential stem cells. Taken together our data suggest that the differences between tumour stem cell and differentiated cell are particular fluent in glioblastoma.

Glucocorticoids attenuate acute graft-versus-host disease by suppressing the cytotoxic capacity of CD8(+) T cells.

Glucocorticoids (GCs) are released from the adrenal gland during inflammation and help to keep immune responses at bay. Owing to their potent anti-inflammatory activity, GCs also play a key role in controlling acute graft-versus-host disease (aGvHD). Here we demonstrate that mice lacking the glucocorticoid receptor (GR) in T cells develop fulminant disease after allogeneic bone marrow transplantation. In a fully MHC-mismatched model, transfer of GR-deficient T cells resulted in severe aGvHD symptoms and strongly decreased survival times. Histopathological features were aggravated and infiltration of CD8(+) T cells into the jejunum was increased when the GR was not expressed. Furthermore, serum levels of IL-2, IFNγ, and IL-17 were elevated and the cytotoxicity of CD8(+) T cells was enhanced after transfer of GR-deficient T cells. Short-term treatment with dexamethasone reduced cytokine secretion but neither impacted disease severity nor the CTLs' cytolytic capacity. Importantly, in an aGvHD model in which disease development exclusively depends on the presence of CD8(+) T cells in the transplant, transfer of GR-deficient T cells aggravated clinical symptoms and reduced survival times as well. Taken together, our findings highlight that suppression of CD8(+) T-cell function is a crucial mechanism in the control of aGvHD by endogenous GCs.

TRAIL-receptor costimulation inhibits proximal TCR signaling and suppresses human T cell activation and proliferation.

The TRAIL-receptor/TRAIL system originally described to induce apoptosis preferentially in malignant cells is also known to be involved in T cell homeostasis and the response to viral infections and autoimmune diseases. Whereas the expression of TRAIL on activated NK and T cells increases their cytotoxicity, induction of TRAIL on APCs can turn them into apoptosis inducers but might also change their immunostimulatory capacity. Therefore, we analyzed how TRAIL-receptor (TRAIL-R) costimulation is modulating TCR-mediated activation of human T cells. T cells triggered by rTRAIL in combination with anti-CD3 and -CD28 Abs exhibited a strong decrease in the expression of activation markers and Th1 and Th2 cytokines compared with CD3/CD28-activated T cells. Most importantly, proliferation of TRAIL-R costimulated T cells was strongly impaired, but no apoptosis was induced. Addition of exogenous IL-2 could not rescue T cells silenced by TRAIL-R costimulation, and TRAIL-mediated inhibition of T cell proliferation only prevented TCR-triggered proliferation but was ineffective if T cells were activated downstream of the TCR. Inhibition of T cell proliferation was associated with abrogation of proximal TCR signaling by inhibiting recruitment of TCR-associated signaling molecules to lipid rafts, followed by abrogation of protein tyrosine phosphorylation of ZAP70, phospholipase C-γ1, and protein kinase C-θ, and impaired nuclear translocation of NFAT, AP-1, and NF-κB. Most importantly, TRAIL-R costimulation efficiently inhibited alloantigen-induced T cell proliferation and CD3/28-induced activation and proliferation of autoreactive T cells derived from patients with Omenn syndrome, indicating that coactivation of TRAIL-R and TCR represents a mechanism to downmodulate T cell immune responses.

Recombinant CD95-Fc (APG101) prevents graft-versus-host disease in mice without disabling antitumor cytotoxicity and T-cell functions.

Graft-versus-host disease (GVHD) induced by transplant-derived T cells represents a major complication after allogeneic bone marrow transplantation (BMT). However, these T cells support engraftment, early T-cell immunity, and mediate the graft-versus-tumor (GVT) effect. Cytotoxic effector functions by transplanted T cells are predominantly mediated by the perforin/granzyme and the CD95/CD95L system. APG101, a novel recombinant human fusion protein consisting of the extracellular domain of CD95 and the Fc domain of an IgG1 antibody inhibited CD95L-induced apoptosis without interfering with T-cell function in vitro and was therefore tested for its ability to prevent GVHD in murine BMT models across minor or major histocompatibility barriers. Starting APG101 treatment either 1 day before or 6 days after transplantation effectively reduced clinical GVHD and rescued survival between 60% and 100% if GVHD was CD95L mediated. APG101 did not interfere with the GVT effect, because P815 mastocytoma and most importantly primary Bcr-Abl-transformed B-cell leukemias were completely eradicated by the alloantigen-specific T cells. Phenotype and homing of alloantigen-specific T cells or their perforin/granzyme-mediated cytotoxicity and proliferative capacity were not affected by APG101 treatment suggesting that APG101 therapy might be useful in GVHD prophylaxis without impairing T-cell function and most importantly preserving GVT activity.

Neutrophil-derived catecholamines mediate negative stress effects on bone.

Mental traumatization is associated with long-bone growth retardation, osteoporosis and increased fracture risk. We revealed earlier that mental trauma disturbs cartilage-to-bone transition during bone growth and repair in mice. Trauma increased tyrosine hydroxylase-expressing neutrophils in bone marrow and fracture callus. Here we show that tyrosine hydroxylase expression in the fracture hematoma of patients correlates positively with acknowledged stress, depression, and pain scores as well as individual ratings of healing-impairment and pain-perception post-fracture. Moreover, mice lacking tyrosine hydroxylase in myeloid cells are protected from chronic psychosocial stress-induced disturbance of bone growth and healing. Chondrocyte-specific ß2-adrenoceptor-deficient mice are also protected from stress-induced bone growth retardation. In summary, our preclinical data identify locally secreted catecholamines in concert with ß2-adrenoceptor signalling in chondrocytes as mediators of negative stress effects on bone growth and repair. Given our clinical data, these mechanistic insights seem to be of strong translational relevance.

Downregulation of FLIP by cycloheximide sensitizes human fat cells to CD95-induced apoptosis.

Adipocyte apoptosis is an important regulator of adipocyte number in fat depots. We have previously shown that an inhibition of protein synthesis sensitizes human adipocytes for apoptosis. In vivo, dramatic changes in the fat cell's protein expression should be anticipated under special conditions such as calorie restriction. Here, we studied the underlying mechanism by which human preadipocytes and adipocytes are sensitized for death receptor induced apoptosis in vitro. The protein synthesis blocker cycloheximide (CHX) sensitized human fat cells for CD95-induced apoptosis in a caspase-dependent manner. Treatment with CHX differentially changed expression of pro- and anti-apoptotic proteins. Most noticeably, FLICE-like inhibitory protein (FLIP) expression rapidly decreased during CHX treatment. Reduction of FLIP levels resulted in undetectable amounts of FLIP at the CD95 death-inducing signaling complex (DISC) upon CD95 stimulation, thereby enhancing recruitment and activation at caspase-8. Down-regulation of FLIP by shRNA sensitized preadipocytes for CD95-induced apoptosis. In mice, adipose tissue mRNA levels of Flip were down-regulated upon fasting. In conclusion, we identify FLIP as an important regulator of apoptosis sensitivity in fat cells. Modulating adipocyte homeostasis by apoptosis might provide a new therapeutic concept to get rid of excess adipose tissue, and FLIP might be a possible target molecule.

Modeling acute graft-versus-host disease (aGVHD) in murine bone marrow transplantation (BMT) models with MHC disparity.

For more than 50years, hematopoietic stem cell transplantation (HSCT) has been the major curative therapy for hematological malignancies and genetic disorders, but its success is limited by the development of graft-versus-host disease (GVHD). GVHD represents a post-transplantation disorder representing the immune-mediated attack of transplant-derived T cells against recipient tissue finally leading to increased morbidity and mortality of the recipient. GVHD develops if donor and recipient are disparate in major or minor histocompatibility antigens (MHC, miHA). Most of the initial knowledge about the biology of GVHD is derived from murine bone marrow transplantation (BMT) models. Of course, GVHD mouse models do not reflect one to one the human situation, but they contribute significantly to our understanding how conditioning and danger signals activate the immune system, enlighten the role of individual molecules, e.g., cytokines, chemokines, death-inducing ligands, define the function of lymphocytes subpopulations for GVHD development and have significant impact on establishing new treatment and prevention strategies used in clinical HSCT. This chapter describes in detail the procedure of allogeneic BMT and the development of GVHD in two commonly used allogeneic murine BMT models (B6→B6.bm1, B6→B6D2F1) with different MHC disparities, which can be used as a basis for advanced studies of GVHD pathology or the development of new treatment strategies.

Adoptively Transferred in vitro-Generated Myeloid-Derived Suppressor Cells Improve T-Cell Function and Antigen-Specific Immunity after Traumatic Lung Injury.

Immune reactions after trauma are characterized by immediate activation of innate immunity and simultaneously downregulation of adaptive immunity leading to a misbalanced immunohomeostasis and immunosuppression of the injured host. Therefore, the susceptibility to secondary infections is strongly increased after trauma. Immune responses are regulated by a network of immune cells influencing each other and at the same time modifying their functions dependent on the inflammatory environment. Although myeloid-derived suppressor cells (MDSCs) are initially described as T-cell suppressors, their immunomodulatory capacity after trauma is mostly undefined. Therefore, in vitro-generated MDSCs were adoptively transferred into mice after blunt chest trauma (TxT). A single MDSC treatment-induced splenic T-cell expansion decreased apoptosis sensitivity and improved proliferation in the absence of T-cell exhaustion until 2 weeks after trauma. MDSC treatment had a long-lasting effect on the genomic landscape of CD4+ T cells by upregulating primarily Th2-associated genes. Remarkably, immune-activating functions of MDSCs supported the ability of TxT mice to respond to post-traumatic secondary antigen challenge. Secondary insults were mimicked by immunizing MDSC-treated TxT mice with ovalbumin (OVA), followed by OVA restimulation in vitro. MDSC treatment significantly increased the frequency of OVA-specific T cells, enhanced their Th1/Th2 cytokine expression, and induced upregulation of cytolytic molecules finally improving OVA-specific cytotoxicity. Overall, we could show that therapeutic MDSC treatment after TxT improves post-traumatic T-cell functions, which might enable the traumatic host to counterbalance trauma-induced immunoparalysis.

Compound heterozygous variants in OTULIN are associated with fulminant atypical late-onset ORAS.

Autoinflammatory diseases are a heterogenous group of disorders defined by fever and systemic inflammation suggesting involvement of genes regulating innate immune responses. Patients with homozygous loss-of-function variants in the OTU-deubiquitinase OTULIN suffer from neonatal-onset OTULIN-related autoinflammatory syndrome (ORAS) characterized by fever, panniculitis, diarrhea, and arthritis. Here, we describe an atypical form of ORAS with distinct clinical manifestation of the disease caused by two new compound heterozygous variants (c.258G>A (p.M86I)/c.500G>C (p.W167S)) in the OTULIN gene in a 7-year-old affected by a life-threatening autoinflammatory episode with sterile abscess formation. On the molecular level, we find binding of OTULIN to linear ubiquitin to be compromised by both variants; however, protein stability and catalytic activity is most affected by OTULIN variant p.W167S. These molecular changes together lead to increased levels of linear ubiquitin linkages in patient-derived cells triggering the disease. Our data indicate that the spectrum of ORAS patients is more diverse than previously thought and, thus, supposedly asymptomatic individuals might also be affected. Based on our results, we propose to subdivide the ORAS into classical and atypical entities.

High antibody levels and reduced cellular response in children up to one year after SARS-CoV-2 infection.

The COVID-19 course and immunity differ in children and adults. We analyzed immune response dynamics in 28 families up to 12 months after mild or asymptomatic infection. Unlike adults, the initial response is plasmablast-driven in children. Four months after infection, children show an enhanced specific antibody response and lower but detectable spike 1 protein (S1)-specific B and T cell responses than their parents. While specific antibodies decline, neutralizing antibody activity and breadth increase in both groups. The frequencies of S1-specific B and T cell responses remain stable. However, in children, one year after infection, an increase in the S1-specific IgA class switch and the expression of CD27 on S1-specific B cells and T cell maturation are observed. These results, together with the enhanced neutralizing potential and breadth of the specific antibodies, suggest a progressive maturation of the S1-specific immune response. Hence, the immune response in children persists over 12 months but dynamically changes in quality, with progressive neutralizing, breadth, and memory maturation. This implies a benefit for booster vaccination in children to consolidate memory formation.

Graft-Versus-Host Disease Prevention by In Vitro-Generated Myeloid-Derived Suppressor Cells Is Exclusively Mediated by the CD11b+CD11c+ MDSC Subpopulation.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid progenitor cells that dampen overwhelming adaptive immune responses through multiple mechanisms and are recognized as an attractive novel immune intervention therapy for counteracting the destructive effects of graft- versus -host disease (GVHD) developing after allogeneic bone marrow transplantation (BMT). MDSCs can be produced in great numbers for cellular therapy, but they present a mixture of subsets whose functions in GVHD prevention are undefined. Here, we generated MDSCs in vitro from murine BM cells in the presence of GM-CSF and defined the integrin CD11c as a marker to subdivide MDSCs into two functional subgroups: CD11b+CD11c+ and CD11b+CD11c- MDSCs. Isolated CD11b+CD11c+ and CD11b+CD11c- MDSCs both inhibited alloantigen-stimulated T-cell proliferation in vitro, although CD11b+CD11c+ MDSCs were more efficient and expressed higher levels of different immunosuppressive molecules. Likewise, expression of surface markers such as MHC class II, CD80, CD86, or PD-L1 further delineated both subsets. Most importantly, only the adoptive transfer of CD11b+CD11c+ MDSCs into a single MHC class I-disparate allogeneic BMT model prevented GVHD development and strongly decreased disease-induced mortality, while CD11b+CD11c- MDSCs were totally ineffective. Surprisingly, allogeneic T-cell homing and expansion in lymphatic and GVHD target organs were not affected by cotransplanted CD11b+CD11c+ MDSCs indicating a clear contradiction between in vitro and in vivo functions of MDSCs. However, CD11b+CD11c+ MDSCs shifted immune responses towards type 2 immunity reflected by increased Th2-specific cytokine expression of allogeneic T cells. Induction of type 2 immunity was mandatory for GVHD prevention, since CD11b+CD11c+ MDSCs were ineffective if recipients were reconstituted with STAT6-deficient T cells unable to differentiate into Th2 cells. Most importantly, the beneficial graft- versus -tumor (GVT) effect was maintained in the presence of CD11b+CD11c+ MDSCs since syngeneic tumor cells were efficiently eradicated. Strong differences in the transcriptomic landscape of both subpopulations underlined their functional differences. Defining CD11b+CD11c+ MDSCs as the subset of in vitro-generated MDSCs able to inhibit GVHD development might help to increase efficiency of MDSC therapy and to further delineate relevant target molecules and signaling pathways responsible for GVHD prevention.

Immunostimulatory functions of adoptively transferred MDSCs in experimental blunt chest trauma.

Myeloid-derived suppressor cells (MDSCs) expand during inflammation and exhibit immunomodulatory functions on innate and adaptive immunity. However, their impact on trauma-induced immune responses, characterized by an early pro-inflammatory phase and dysregulated adaptive immunity involving lymphocyte apoptosis, exhaustion and unresponsiveness is less clear. Therefore, we adoptively transferred in vitro-generated MDSCs shortly before experimental blunt chest trauma (TxT). MDSCs preferentially homed into spleen and liver, but were undetectable in the injured lung, although pro-inflammatory mediators transiently increased in the bronchoalveolar lavage (BAL). Surprisingly, MDSC treatment strongly increased splenocyte numbers, however, without altering the percentage of splenic leukocyte populations. T cells of MDSC-treated TxT mice exhibited an activated phenotype characterized by expression of activation markers and elevated proliferative capacity in vitro, which was not accompanied by up-regulated exhaustion markers or unresponsiveness towards in vitro activation. Most importantly, also T cell expansion after staphylococcal enterotoxin B (SEB) stimulation in vivo was unchanged between MDSC-treated or untreated mice. After MDSC transfer, T cells preferentially exhibited a Th1 phenotype, a prerequisite to circumvent post-traumatic infectious complications. Our findings reveal a totally unexpected immunostimulatory role of adoptively transferred MDSCs in TxT and might offer options to interfere with post-traumatic malfunction of the adaptive immune response.

Preserved in vitro immunoreactivity in children receiving long-term immunosuppressive therapy due to inflammatory bowel disease or autoimmune hepatitis.

BACKGROUND: Children with inflammatory bowel disease (IBD) or autoimmune hepatitis (AIH) are at risk for severe infections. This is partially a result of their chronic disease condition but, moreover, a side effect of their immunosuppressive therapy. Currently, vaccinations with live vaccines are regarded as contraindicated under immunosuppressive therapy, mainly because of concerns about side effects and a lack of data showing an adequate immune reaction. As there is no systematic study on the individual immunoreactivity under immunosuppressive therapy in this patient group, we analyzed the lymphocyte subgroups and immunoreactivity of lymphocytes in children with IBD or AIH with and without immunosuppressive therapy in vitro. METHODS: We collected whole blood samples from 17 children with IBD or AIH on high-level immunosuppression (IS) (group 1) and 8 on low-level IS (group 2) in comparison with 6 patients without systemic IS (group 3). After Ficoll separation of peripheral mononuclear cells, the samples were analyzed by flow cytometry to determine the lymphocyte subgroups. Furthermore, we stimulated the isolated lymphocytes with phytohemagglutinin (PHA), tetanus antigen, and adenovirus antigen and measured their proliferation by incorporation of H3-thymidine detected in a beta counter. The statistical evaluation was performed by Kruskal-Wallis test and Mann-Whitney U test using a bilateral level of significance of α = 5%. RESULTS: Patients with low- or high-level IS showed no significant difference in the number of lymphocytes or T cells. Interestingly, IS did not influence the lymphocyte proliferation assay significantly regarding median reaction to PHA, tetanus antigen, or adenovirus antigen between the three groups. However, comparing all immunosuppressed patients to the patients without IS, there was a significant difference towards stimulation with tetanus antigen. CONCLUSIONS: Contrary to expectations of a strong influence of IS therapy on the immunoreactivity, this study showed only minor differences between the groups with high-level, low-level, and no IS. Particularly, the in vitro reactivity to adenovirus antigen was nearly the same in all three groups. We assume that-provided a normal distribution and count of lymphocyte subgroups-patients with moderate immunosuppression might be capable of raising an effective immune response to inactivated and live vaccines.

Pharmacological inhibition of IL-6 trans-signaling improves compromised fracture healing after severe trauma.

Patients with multiple injuries frequently suffer bone fractures and are at high risk to develop fracture healing complications. Because of its key role both in systemic posttraumatic inflammation and fracture healing, the pleiotropic cytokine interleukin-6 (IL-6) may be involved in the pathomechanisms of trauma-induced compromised fracture healing. IL-6 signals are transmitted by two different mechanisms: classic signaling via the membrane-bound receptor (mIL-6R) and trans-signaling via its soluble form (sIL-6R). Herein, we investigated whether IL-6 classic and trans-signaling play different roles in bone regeneration after severe injury. Twelve-week-old C57BL/6J mice underwent combined femur osteotomy and thoracic trauma. To study the function of IL-6, either an anti-IL-6 antibody, which inhibits both IL-6 classic and trans-signaling, or a soluble glycoprotein 130 fusion protein (sgp130Fc), which selectively blocks trans-signaling, were injected 30 min and 48 h after surgery. Bone healing was assessed using cytokine analyses, flow cytometry, histology, micro-computed tomography, and biomechanical testing. Selective inhibition of IL-6 trans-signaling significantly improved the fracture healing outcome after combined injury, as confirmed by accelerated cartilage-to-bone transformation, enhanced bony bridging of the fracture gap and improved mechanical callus properties. In contrast, global IL-6 inhibition did not affect compromised fracture healing. These data suggest that classic signaling may mediate beneficial effects on bone repair after severe injury. Selective inhibition of IL-6 trans-signaling might have therapeutic potential to treat fracture healing complications in patients with concomitant injuries.