LAG-3 Inhibitory Receptor Expression Identifies Immunosuppressive Natural Regulatory Plasma Cells.

B lymphocytes can suppress immunity through interleukin (IL)-10 production in infectious, autoimmune, and malignant diseases. Here, we have identified a natural plasma cell subset that distinctively expresses the inhibitory receptor LAG-3 and mediates this function in vivo. These plasma cells also express the inhibitory receptors CD200, PD-L1, and PD-L2. They develop from various B cell subsets in a B cell receptor (BCR)-dependent manner independently of microbiota in naive mice. After challenge they upregulate IL-10 expression via a Toll-like receptor-driven mechanism within hours and without proliferating. This function is associated with a unique transcriptome and epigenome, including the lowest amount of DNA methylation at the Il10 locus compared to other B cell subsets. Their augmented accumulation in naive mutant mice with increased BCR signaling correlates with the inhibition of memory T cell formation and vaccine efficacy after challenge. These natural regulatory plasma cells may be of broad relevance for disease intervention.

Development of epithelial cholinergic chemosensory cells of the urethra and trachea of mice.

Cholinergic chemosensory cells (CCC) are infrequent epithelial cells with immunosensor function, positioned in mucosal epithelia preferentially near body entry sites in mammals including man. Given their adaptive capacity in response to infection and their role in combatting pathogens, we here addressed the time points of their initial emergence as well as their postnatal development from first exposure to environmental microbiota (i.e., birth) to adulthood in urethra and trachea, utilizing choline acetyltransferase (ChAT)-eGFP reporter mice, mice with genetic deletion of MyD88, toll-like receptor-2 (TLR2), TLR4, TLR2/TLR4, and germ-free mice. Appearance of CCC differs between the investigated organs. CCC of the trachea emerge during embryonic development at E18 and expand further after birth. Urethral CCC show gender diversity and appear first at P6-P10 in male and at P11-P20 in female mice. Urethrae and tracheae of MyD88- and TLR-deficient mice showed significantly fewer CCC in all four investigated deficient strains, with the effect being most prominent in the urethra. In germ-free mice, however, CCC numbers were not reduced, indicating that TLR2/4-MyD88 signaling, but not vita-PAMPs, governs CCC development. Collectively, our data show a marked postnatal expansion of CCC populations with distinct organ-specific features, including the relative impact of TLR2/4-MyD88 signaling. Strong dependency on this pathway (urethra) correlates with absence of CCC at birth and gender-specific initial development and expansion dynamics, whereas moderate dependency (trachea) coincides with presence of first CCC at E18 and sex-independent further development.

Physical exercise induces specific adaptations resulting in reduced organ injury and mortality during severe polymicrobial sepsis.

OBJECTIVES: High physical activity levels are associated with wide-ranging health benefits, disease prevention, and longevity. In the present study, we examined the impact of regular physical exercise on the severity of organ injury and survival probability, as well as characteristics of the systemic immune and metabolic response during severe polymicrobial sepsis. DESIGN: Animal study. SETTING: University laboratory. SUBJECTS: Male C57BL/6N mice. INTERVENTIONS: Mice were trained for 6 weeks by treadmill and voluntary wheel running or housed normally. Polymicrobial sepsis in mice was induced by injection of fecal slurry. Subsequently, mice were randomized into the following groups: healthy controls, 6 hours postsepsis, and 24 hours postsepsis. MEASUREMENTS AND MAIN RESULTS: Blood and organ samples were collected and investigated by measuring clinical chemistry variables, cytokines, plasma metabolites, and bacterial clearance. Organ morphology and damage were characterized by histological staining. Physical exercise improved survival and the ability of bacterial clearance in blood and organs. The release of pro- and anti-inflammatory cytokines, including interleukin-6 and interleukin-10, was diminished in trained compared to untrained mice during sepsis. The sepsis-associated acute kidney tubular damage was less pronounced in pretrained animals. By metabolic profiling and regression analysis, we detected lysophosphatidylcholine 14:0, tryptophan, as well as pimelylcarnitine linked with levels of neutrophil gelatinase-associated lipocalin representing acute tubular injury (corrected R=0.910; p<0.001). We identified plasma lysophosphatidylcholine 16:0, lysophosphatidylcholine 17:0, and lysophosphatidylcholine 18:0 as significant metabolites discriminating between trained and untrained mice during sepsis. CONCLUSIONS: Regular physical exercise reduces sepsis-associated acute kidney injury and death. As a specific mechanism of exercise-induced adaptation, we identified various lysophosphatidylcholines that might function as surrogate for improved outcome in sepsis.

Distinct different contributions of the alternative and classical complement activation pathway for the innate host response during sepsis.

Complement activation represents a crucial innate defense mechanism to invading microorganisms, but there is an eminent lack of understanding of the separate contribution of the different complement activation pathways to the host response during sepsis. We therefore investigated different innate host immune responses during cecal ligation and puncture (CLP)-induced sepsis in mice lacking either the alternative (fD(-/-)) or classical (C1q(-/-)) complement activation pathway. Both knockout mice strains showed a significantly reduced survival and increased organ dysfunction when compared with control mice. Surprisingly, fD(-/-) mice demonstrated a compensated bacterial clearance capacity as control mice at 6 h post CLP, whereas C1q(-/-) mice were already overwhelmed by bacterial growth at this time point. Interestingly, at 24 h after CLP, fD(-/-) mice failed to clear bacteria in a way comparable to control mice. However, both knockout mice strains showed compromised C3 cleavage during sepsis. Investigating potential causes for this discrepancy, we were able to demonstrate that despite normal bacterial clearance capacity early during the onset of sepsis, fD(-/-) mice displayed increased inflammatory cytokine generation and neutrophil recruitment into lungs and blood when compared with both control- and C1q(-/-) mice, indicating a potential loss of control over these immune responses. Further in vitro experiments revealed a strongly increased Nf-κB activation capacity in isolated neutrophils from fD(-/-) mice, supporting this hypothesis. Our results provide evidence for the new concept that the alternative complement activation pathway exerts a distinctly different contribution to the innate host response during sepsis when compared with the classical pathway.

Visualization of Inflammation in Experimental Colitis by Magnetic Resonance Imaging Using Very Small Superparamagnetic Iron Oxide Particles.

Inflammatory bowel diseases (IBD) comprise mainly ulcerative colitis (UC) and Crohn´s disease (CD). Both forms present with a chronic inflammation of the (gastro) intestinal tract, which induces excessive changes in the composition of the associated extracellular matrix (ECM). In UC, the inflammation is limited to the colon, whereas it can occur throughout the entire gastrointestinal tract in CD. Tools for early diagnosis of IBD are still very limited and highly invasive and measures for standardized evaluation of structural changes are scarce. To investigate an efficient non-invasive way of diagnosing intestinal inflammation and early changes of the ECM, very small superparamagnetic iron oxide nanoparticles (VSOPs) in magnetic resonance imaging (MRI) were applied in two mouse models of experimental colitis: the dextran sulfate sodium (DSS)-induced colitis and the transfer model of colitis. For further validation of ECM changes and inflammation, tissue sections were analyzed by immunohistochemistry. For in depth ex-vivo investigation of VSOPs localization within the tissue, Europium-doped VSOPs served to visualize the contrast agent by imaging mass cytometry (IMC). VSOPs accumulation in the inflamed colon wall of DSS-induced colitis mice was visualized in T2* weighted MRI scans. Components of the ECM, especially the hyaluronic acid content, were found to influence VSOPs binding. Using IMC, co-localization of VSOPs with macrophages and endothelial cells in colon tissue was shown. In contrast to the DSS model, colonic inflammation could not be visualized with VSOP-enhanced MRI in transfer colitis. VSOPs present a potential contrast agent for contrast-enhanced MRI to detect intestinal inflammation in mice at an early stage and in a less invasive manner depending on hyaluronic acid content.

Characteristics of clinical sepsis reflected in a reliable and reproducible rodent sepsis model.

BACKGROUND: Sepsis models are frequently based on induction of peritonitis, with cecal ligation and puncture reflecting the prototypical model. However, there is an ongoing discussion about the limitations of these models due to their variability in progression and outcome. Since standardization is a cornerstone of experimental models, we aimed to develop a reliable and reproducible procedure for induction of peritonitis. MATERIALS AND METHODS: A human stool batch was processed for -80° storage. For induction of peritonitis in fluid-resuscitated rats, a defined volume of stool suspension from this batch was injected intraperitoneally. For characterization of the model, physiologic and inflammatory changes were evaluated after sepsis induction. Survival analyses with the same batch were repeated in four independent experiments over a time period of 16 mo. RESULTS: The polymicrobial infection resulted in severe peritoneal inflammation with a systemic increase in cytokines. The mortality rate at 15 h was 29% and this was reproducible over a 16 mo time period. If antibiotic treatment was applied, a 50% survival was achieved. Laboratory markers indicated a progressive multi-organ dysfunction, while blood gas analysis showed respiratory compensation of a metabolic acidosis, and maintenance of PaO(2). Intravital microscopy of the liver revealed an impaired microcirculation. A decreased hemostatic potential was demonstrated by rotational thromboelastometry. Despite clinical recovery within 3 d, surviving animals showed laboratory and histologic signs of persisting inflammation even after 2 wk. CONCLUSIONS: This model reflects many features of human sepsis. Application of an infectious focus that is both quantitatively and qualitatively defined assures high reproducibility. Moreover, the procedure is simple and can be easily standardized.

Cardioprotective effect of NO-metoprolol in murine coxsackievirus B3-induced myocarditis.

The effect of NO-metoprolol, that is, 3-nitrooxypivaloyl metoprolol-amide, a novel NO-releasing derivative of the ß1-blocking drug metoprolol was investigated in A.CA/SnJ mice infected with coxsackievirus B3 (CVB3) and compared to metoprolol and placebo. Daily treatment of mice with the respective drug started immediately (experiment A) or 3 days after virus infection (experiment B) and was continued until day 13 post-infection (p.i.). Two doses of NO-metoprolol were administered. Body mass differences, viral load, and histopathological signs of myocarditis were compared between the several groups. As a result, NO-metoprolol diminished significantly the body weight loss, the viral load and the histopathology, whereas metoprolol treatment led solely to a significant attenuation of myocardial damage. In experiment A, low dose NO-metoprolol decreased significantly enteroviral copy numbers. Both doses of NO-metoprolol had a significant effect on reduction of myocardial infiltrates and fibrosis. The data suggest that delayed drug administration might more advantageous. Both doses of NO-metoprolol reduced significantly the scores of four tested parameters compared to placebo. Body weight loss, virus titers, plus-strand as well as minus-strand enteroviral RNA levels, infiltration and fibrosis scores were diminished significantly when NO-metoprolol was given 3 days p.i. In addition, a significant difference regarding the enteroviral copy numbers was observed between low dose NO-metoprolol- and metoprolol-treated mice. Treatment with metoprolol reduced insignificantly the viral load and body weight loss (experiment A and B) but led to a significant reduction of myocardial histopathology in experiment A. The results indicate that NO-metoprolol treatment has a greater therapeutic benefit than metoprolol.

Akkermansia muciniphila strain ATCC BAA-835 does not promote short-term intestinal inflammation in gnotobiotic interleukin-10-deficient mice.

Akkermansia muciniphila is a common member of the intestinal microbiota of healthy human individuals. Its abundance is negatively associated with inflammatory bowel disease and metabolic disorders and the oral administration of A. muciniphila improves the symptoms of metabolic disease in mice. Therefore, A. muciniphila is a promising candidate for the treatment of type-2 diabetes and obesity. However, some studies using animal models of intestinal inflammation reported that A. muciniphila may exacerbate gut inflammation. Because of these contradictory reports the present study aimed to clarify the role of A. muciniphila in the development of intestinal inflammation and the conditions promoting it. For this purpose, the short-term colitogenic potential of A. muciniphila strain ATCC BAA-835 was investigated in colitis-prone, gnotobiotic IL-10-deficient (Il10-/-) mice. Il10-/- mice mono-associated with A. muciniphila showed no signs of intestinal inflammation based on body-weight change, histopathological scoring and inflammatory markers. Additional association of the mice with the colitogenic Escherichia coli strain NC101 led to cecal but not colonic inflammation. However, the severity of the inflammation did not exceed that observed in mice mono-associated with E. coli NC101. Il10-/- mice colonized with a simplified human intestinal microbiota showed increased histopathology, but no increase in inflammatory markers. Furthermore, co-colonization with A. muciniphila did not modify histopathology. The turnover of intestinal mucus was similar in all groups despite the mucus-degrading property of A. muciniphila. Overall, the data do not support a short-term pro-inflammatory effect of A. muciniphila strain ATCC BAA-835 in the Il10-/- mouse model for inflammatory bowel disease.

Dysfunctional high-density lipoprotein activates toll-like receptors via serum amyloid A in vascular smooth muscle cells.

Serum amyloid A (SAA) is an uremic toxin and acute phase protein. It accumulates under inflammatory conditions associated with high cardiovascular morbidity and mortality in patients with sepsis or end-stage renal disease (ESRD). SAA is an apolipoprotein of the high-density lipoprotein (HDL). SAA accumulation turns HDL from an anti-inflammatory to a pro-inflammatory particle. SAA activates monocyte chemoattractant protein-1 (MCP-1) in vascular smooth muscle cells. However, the SAA receptor-mediated signaling pathway in vascular cells is poorly understood. Therefore, the SAA-mediated signaling pathway for MCP-1 production was investigated in this study. The SAA-induced MCP-1 production is dependent on the activation of TLR2 and TLR4 as determined by studies with specific receptor antagonists and agonists or siRNA approach. Experiments were confirmed in tissues from TLR2 knockout, TLR4 deficient and TLR2 knock-out/TLR4 deficient mice. The intracellular signaling pathway is IκBα and subsequently NFκB dependent. The MCP-1 production induced by SAA-enriched HDL and HDL isolated from septic patients with high SAA content is also TLR2 and TLR4 dependent. Taken together, the TLR2 and TLR4 receptors are functional SAA receptors mediating MCP-1 release. Furthermore, the TLR2 and TLR4 are receptors for dysfunctional HDL. These results give a further inside in SAA as uremic toxin involved in uremia-related pro-inflammatory response in the vascular wall.

Microbial Colonization in Adulthood Shapes the Intestinal Macrophage Compartment.

BACKGROUND AND AIMS: Contact with distinct microbiota early in life has been shown to educate the mucosal immune system, hence providing protection against immune-mediated diseases. However, the impact of early versus late colonization with regard to the development of the intestinal macrophage compartment has not been studied so far. METHODS: Germ-free mice were colonized with specific-pathogen-free [SPF] microbiota at the age of 5 weeks. The ileal and colonic macrophage compartment were analysed by immunohistochemistry, flow cytometry, and RNA sequencing 1 and 5 weeks after colonization and in age-matched SPF mice, which had had contact with microbiota since birth. To evaluate the functional differences, dextran sulfate sodium [DSS]-induced colitis was induced, and barrier function analyses were undertaken. RESULTS: Germ-free mice were characterized by an atrophied intestinal wall and a profoundly reduced number of ileal macrophages. Strikingly, morphological restoration of the intestine occurred within the first week after colonization. In contrast, ileal macrophages required 5 weeks for complete restoration, whereas colonic macrophages were numerically unaffected. However, following DSS exposure, the presence of microbiota was a prerequisite for colonic macrophage infiltration. One week after colonization, mild colonic inflammation was observed, paralleled by a reduced inflammatory response after DSS treatment, in comparison with SPF mice. This attenuated inflammation was paralleled by a lack of TNFα production of LPS-stimulated colonic macrophages from SPF and colonized mice, suggesting desensitization of colonized mice by the colonization itself. CONCLUSIONS: This study provides the first data indicating that after colonization of adult mice, the numeric, phenotypic, and functional restoration of the macrophage compartment requires the presence of intestinal microbiota and is time dependent.

Comparative suitability of CFDA-SE and rhodamine 6G for in vivo assessment of leukocyte-endothelium interactions.

Intravital fluorescence microscopy (IVM) is a predestined tool for investigating the fate of leukocytes during the process of leukocyte recruitment. In the present study, the commonly used dye for this purpose, rhodamine 6G, and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) were compared for leukocytes labelling with respect to suitability for IVM studies. Their potential in labelling different leukocytes subpopulations as well as their fluorescence intensities were assessed by flow cytometry revealing distinct differences between both dyes. These differences had a profound impact on their application for in vivo imaging of leukocyte-endothelium interactions. In summary, CFDA-SE revealed superior in labelling leukocytes for in vivo microscopy with respect to image quality. In addition, we could show the efficiency of CFDA-SE also under disease condition in an animal model of sepsis.

Assessment of rat antigen-induced arthritis and its suppression during glucocorticoid therapy by use of hemicyanine dye probes with different molecular weight in near-infrared fluorescence optical imaging.

PURPOSE: Arthritic joints are ideal disease entities to be assessed via optical imaging. Here, we investigated the selective accumulation behavior of two differently sized hemicyanine optical probes in arthritic joints and its modification during glucocorticoid therapy in the course of inflammation. MATERIALS AND METHODS: The well-standardized preclinical antigen-induced arthritis (AIA) model in rats was used. The animals were divided into 3 groups: arthritic, arthritic and dexamethasone-treated, and immunized only. After intravenous coinjection of DY-752 (size, 800 Da) and DY-682-(rat) IgG (size, 150 kDa) probes, spectrally unmixed near-infrared fluorescence images were acquired and analyzed semiquantitatively. Probe organ distribution, joint swelling, blood cell counts, joint vessel density, and histological scoring of arthritis were determined. RESULTS: The local joint accumulation kinetics of the DY-752 probe differed from the DY-682-IgG one. In the course of AIA, probe signaling in arthritic joints was similar between each other. Joint swelling and histological scoring were in accordance with signaling. Dexamethasone treatment of rats with AIA significantly reduced both the near-infrared fluorescence signals and severity of arthritis but did not change the joint vascular density or the uptake of the probes by phagocytes. A differential biodistribution of both probes was encountered, but such an accumulation was prevented by dexamethasone treatment. CONCLUSIONS: Near-infrared fluorescence signaling in the course of AIA closely reflects the pathophysiological events of the arthritic joint and the effects of therapy independently of the molecular size of the probe. The results show the suitability of our hemicyanine probes for imaging of arthritis.

Role of p38 mitogen-activated protein kinase in posttraumatic immunosuppression in mice.

BACKGROUND: Patients with multiple injuries surviving the initial insult are highly susceptible to secondary pneumonia, frequently progressing into sepsis and multiorgan failure. However, the underlying mechanisms of posttraumatic immunosuppression are poorly understood. We hypothesized that dysregulated p38 mitogen-activated protein kinase (MAPK) signaling accounts for impaired lung protective immunity in a model of trauma/hemorrhage (T/H) and subsequent pneumococcal pneumonia in mice. METHODS: C57BL6/N mice were subjected to trauma by midline laparotomy, and T/H was induced by midline laparotomy followed by cannulation of femoral arteries and veins to induce hemorrhage. Subsequently, mice were infected with Streptococcus pneumoniae. In selected experiments, mice were treated with a p38 MAPK inhibitor or vehicle control immediately after induction of T/H. RESULTS: Mice subjected to T/H showed significantly increased p38 MAPK activation in their lungs, which was accompanied by a reduced Escherichia coli phagocytosis by macrophages from T/H mice in vitro and an impaired pneumococcal killing activity of T/H mice in vivo, overall resulting in increased mortality of T/H mice after infection with S. pneumoniae. Application of p38 MAPK inhibitor BIRB796 immediately after T/H induction improved the bacterial phagocytosis activity of macrophages from T/H mice in vitro and lung pneumococcal killing in vivo but did not improve the survival of T/H mice challenged with S. pneumoniae. CONCLUSION: T/H triggers sustained p38 MAPK activation in the lungs of mice, which attenuates lung macrophage antibacterial activities and renders mice more susceptible to pneumococcal pneumonia. However, no major role for dysregulated p38 MAPK to affect survival of T/H mice after pneumococcal challenge was detected, suggesting that dysregulated p38 MAPK activity may possibly play only a limited role in posttraumatic immunosuppression in mice.