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Abstract Malnutrition is linked to 45% of global childhood mortality, however, the impact of maternal malnutrition on the child’s health remains elusive. Previous studies suggested that maternal malnutrition does not affect breast milk composition. Yet, malnourished children often develop a so-called environmental enteropathy, assumed to be triggered by frequent pathogen uptake and unfavorable gut colonization. Here, we show in a murine model that maternal malnutrition induces a persistent inflammatory gut dysfunction in the offspring that establishes during nursing and does not recover after weaning onto standard diet. Early intestinal influx of neutrophils, impaired postnatal development of gut-regulatory functions, and expansion of Enterobacteriaceae were hallmarks of this enteropathy. This gut phenotype resembled those developing under deficient S100a8/a9-supply via breast milk, which is a known key factor for the postnatal development of gut homeostasis. We could confirm that S100a8/a9 is lacking in the breast milk of malnourished mothers and the offspring’s intestine. Nutritional supply of S100a8 to neonates of malnourished mothers abrogated the aberrant development of gut mucosal immunity and microbiota colonization and protected them lifelong against severe enteric infections and non-infectious bowel diseases. S100a8 supplementation after birth might be a promising measure to counteract deleterious imprinting of gut immunity by maternal malnutrition.

Abstract It has been increasingly recognized that there is a subset of patients with refractory systemic JIA, who have failed all available medications and may benefit from HSCT. The increasing experience with HSCT in SJIA, suggests that despite the complicated post-HSCT course, short-term, the transplanted patients either achieved SJIA remission or reduced burden of disease. Longer follow-up, however, is needed to better define the long-term outcomes. The discussion at the NextGen 2022 conference was focused on the optimal timing for the procedure, the need for a good control of inflammatory SJIA activity prior to HSCT, and the role of the reduced intensity conditioning regimens as there was a remote concern that such regimens might increase the risk of SJIA relapse after the transplantation. There was unanimous agreement about the importance of long-term registries to address these questions.

Introduction Sepsis remains the major cause of death among hospitalised patients in intensive care. While targeting sepsis-causing pathogens with source control or antimicrobials has had a dramatic impact on morbidity and mortality of sepsis patients, this strategy remains insufficient for about one-third of the affected individuals who succumb. Pharmacological targeting of mechanisms that reduce sepsis-defining organ dysfunction may be beneficial. When given at low doses, the anthracycline epirubicin promotes tissue damage control and lessens the severity of sepsis independently of the host–pathogen load by conferring disease tolerance to infection. Since epirubicin at higher doses can be myelotoxic, a first dose–response trial is necessary to assess the potential harm of this drug in this new indication.Methods and analysis Epirubicin for the Treatment of Sepsis and Septic Shock-1 is a randomised, double-blind, placebo-controlled phase 2 dose-escalation phase IIa clinical trial to assess the safety of epirubicin as an adjunctive in patients with sepsis. The primary endpoint is the 14-day myelotoxicity. Secondary and explorative outcomes include 30-day and 90-day mortality, organ dysfunction, pharmacokinetic/pharmacodynamic (PK/PD) and cytokine release. Patients will be randomised in three consecutive phases. For each study phase, patients are randomised to one of the two study arms (epirubicin or placebo) in a 4:1 ratio. Approximately 45 patients will be recruited. Patients in the epirubicin group will receive a single dose of epirubicin (3.75, 7.5 or 15 mg/m2 depending on the study phase. After each study phase, a data and safety monitoring board will recommend continuation or premature stopping of the trial. The primary analyses for each dose level will report the proportion of myelotoxicity together with a 95% CI. A potential dose-toxicity association will be analysed using a logistic regression model with dose as a covariate. All further analyses will be descriptive.Ethics and dissemination The protocol is approved by the German Federal Institute for Drugs and Medical Devices. The results will be submitted for publication in peer-reviewed journals.Trial registration number NCT05033808.

Abstract The heterogeneity of the SARS-CoV-2 immune responses has become considerably more complex over time and diverse immune imprinting is observed in vaccinated individuals. Despite vaccination, following the emergence of the Omicron variant, some individuals appear more susceptible to primary infections and reinfections than others, underscoring the need to elucidate how immune responses are influenced by previous infections and vaccination. IgG, IgA, neutralizing antibodies and T-cell immune responses in 1,325 individuals (955 of which were infection-naive) were investigated before and after three doses of the BNT162b2 vaccine, examining their relation to breakthrough infections and immune imprinting in the context of Omicron. Our study shows that both humoral and cellular responses following vaccination were generally higher after SARS-CoV-2 infection compared to infection-naive. Notably, viral exposure before vaccination was crucial to achieving a robust IgA response. Individuals with lower IgG, IgA, and neutralizing antibody responses postvaccination had a significantly higher risk of reinfection and future Omicron infections. This was not observed for T-cell responses. A primary infection before Omicron and subsequent reinfection with Omicron dampened the humoral and cellular responses compared to a primary Omicron infection, consistent with immune imprinting. These results underscore the significant impact of hybrid immunity for immune responses in general, particularly for IgA responses even after revaccination, and the importance of robust humoral responses in preventing future infections.

Abstract Background With metabolic alterations of the tumor microenvironment (TME) contributing to cancer progression, metastatic spread and response to targeted therapies, non-invasive and repetitive imaging of tumor metabolism is of major importance. The purpose of this study was to investigate whether multiparametric chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI) allows to detect differences in the metabolic profiles of the TME in murine breast cancer models with divergent degrees of malignancy and to assess their response to immunotherapy. Methods Tumor characteristics of highly malignant 4T1 and low malignant 67NR murine breast cancer models were investigated, and their changes during tumor progression and immune checkpoint inhibitor (ICI) treatment were evaluated. For simultaneous analysis of different metabolites, multiparametric CEST-MRI with calculation of asymmetric magnetization transfer ratio (MTRasym) at 1.2 to 2.0 ppm for glucose-weighted, 2.0 ppm for creatine-weighted and 3.2 to 3.6 ppm for amide proton transfer- (APT-) weighted CEST contrast was conducted. Ex vivo validation of MRI results was achieved by 1H nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry imaging with laser postionization and immunohistochemistry. Results During tumor progression, the two tumor models showed divergent trends for all examined CEST contrasts: While glucose- and APT-weighted CEST contrast decreased and creatine-weighted CEST contrast increased over time in the 4T1 model, 67NR tumors exhibited increased glucose- and APT-weighted CEST contrast during disease progression, accompanied by decreased creatine-weighted CEST contrast. Already three days after treatment initiation, CEST contrasts captured response to ICI therapy in both tumor models. Conclusion Multiparametric CEST-MRI enables non-invasive assessment of metabolic signatures of the TME, allowing both for estimation of the degree of tumor malignancy and for assessment of early response to immune checkpoint inhibition.

ABSTRACT The prediction of the durability of immunity against COVID-19 is relevant, and longitudinal studies are essential for unraveling the details regarding protective SARS‐CoV‐2 antibody responses. It has become challenging to discriminate between COVID-19 vaccine- and infection-induced immune responses since all approved vaccines in Europe and the USA are based on the viral spike (S) protein, which is also the most commonly used antigen in immunoassays measuring immunoglobulins (Igs) against SARS-CoV-2. We have developed a nucleocapsid (N) protein-based sandwich ELISA for detecting pan anti-SARS-CoV-2 Ig with a sensitivity and specificity of 97%. Generalized mixed models were used to determine the degree of long‐term humoral immunity against the N protein and the receptor-binding domain (RBD) of the S protein in a cohort of infected individuals to distinguish between COVID-19 vaccine- and infection-induced immunity. N-specific waning could be observed in individuals who did not experience reinfection, while individuals who experienced reinfection had a new significant increase in N-specific Ig levels. In individuals that seroconverted without a reinfection, 70.1% remained anti-N seropositive after 550 days. The anti-RBD Ig dynamics were unaffected by reinfection but exhibited a clear increase in RBD-specific Ig when vaccination was initiated. In conclusion, a clear difference in the dynamics of the antibody response against N protein and RBD was observed over time. Anti-N protein-specific Igs can be detected up to 18 months after SARS-CoV-2 infection allowing long-term discrimination of infectious and vaccine antibody responses. IMPORTANCE Longitudinal studies are essential to unravel details regarding the protective antibody responses after COVID-19 infection and vaccination. It has become challenging to distinguish long-term immune responses to SARS-CoV-2 infection and vaccination since most approved vaccines are based on the viral spike (S) protein, which is also mostly used in immunoassays measuring immunoglobulins (Igs) against SARS-CoV-2. We have developed a novel nucleocapsid (N) protein-based sandwich ELISA for detecting pan-anti-SARS-CoV-2 Ig, exhibiting high sensitivity and specificity. Generalized mixed models were used to determine long‐term humoral immunity in a cohort of infected individuals from the Faroe Islands, distinguishing between COVID-19 vaccine- and infection-induced immunity. A clear difference in the dynamics of the antibody response against N protein and S protein was observed over time, and the anti-N protein-specific Igs could be detected up to 18 months after SARS-CoV-2 infection. This enables long-term discrimination between natural infection and vaccine-dependent antibody responses.

HIV-1 eradication is hindered by viral persistence in different cell reservoirs, including circulatory CD4+ T-cells and tissue-resident macrophages. Here, by analyzing male genital mucosa from cART-suppressed HIV1-infected individuals, Real et al. show that M4 macrophages represent the major macrophage HIV-1 reservoir in this tissue. These macrophages have an inflammatory IL1R+S100A8+MMP7+M4-phenotype, and contain transcriptionally active HIV-1, which reactivate infectious virus production from viral latency in response to autocrine/paracrine S100A8-mediated glycolysis.

Summary: Immune-suppressive effects of myeloid-derived suppressor cells (MDSCs) are well characterized during anti-tumor immunity. The complex mechanisms promoting MDSC development and their regulatory effects during autoimmune diseases are less understood. We demonstrate that the endogenous alarmin S100A8/A9 reprograms myeloid cells to a T cell suppressing phenotype during autoimmune arthritis. Treatment of myeloid precursors with S100-alarmins during differentiation induces MDSCs in a Toll-like receptor 4-dependent manner. Consequently, knockout of S100A8/A9 aggravates disease activity in collagen-induced arthritis due to a deficit of MDSCs in local lymph nodes, which could be corrected by adoptive transfer of S100-induced MDSCs. Blockade of MDSC function in vivo aggravates disease severity in arthritis. Therapeutic application of S100A8 induces MDSCs in vivo and suppresses the inflammatory phenotype of S100A9ko mice. Accordingly, the interplay of T cell-mediated autoimmunity with a defective innate immune regulation is crucial for autoimmune arthritis, which should be considered for future innovative therapeutic options.

Excess ketogenesis can lead to ketoacidosis, a serious complication in patients with diabetes. Here the authors report an insulin independent pathway, the hepatic nonparenchymal S100A9-TLR4-mTORC1 axis, that is able to normalize diabetic ketogenesis and pre-clinical data to suggest potential for development of S100A9 based adjunctive therapy to insulin.

The S100A8/A9 heterocomplex is an abundant damage-associated molecular pattern and mainly expressed by monocytes, inflammatory activated keratinocytes and neutrophilic granulocytes. The heterocomplex as well as the heterotetramer are involved in a variety of diseases and tumorous processes. However, their detailed mode of action and especially which receptors are involved hereby remains to be fully revealed. Several cell surface receptors are reported to interact with S100A8 and/or S100A9, the best studied being the pattern recognition receptor TLR4. RAGE, CD33, CD68, CD69, and CD147, all of them are involved as receptors in various inflammatory processes, are also among these putative binding partners for S100A8 and S100A9. Interactions between S100 proteins and these receptors described so far come from a wide variety of cell culture systems but their biological relevance in vivo for the inflammatory response of myeloid immune cells is not yet clear. In this study, we compared the effect of CRISPR/Cas9 mediated targeted deletion of CD33, CD68, CD69, and CD147 in ER-Hoxb8 monocytes on S100A8 or S100A9 induced cytokine release with TLR4 knockout monocytes. Whereas deletion of TLR4 abolished the S100-induced inflammatory response in monocyte stimulation experiments with both S100A8 and S100A9, knockouts of CD33, CD68, CD69, or CD147 revealed no effect on the cytokine response in monocytes. Thus, TLR4 is the dominant receptor for S100-triggered inflammatory activation of monocytes.

This study investigates the dynamics of immunological markers after first SARS-CoV-2 vaccination dose in cohort of healthcare professionals in Denmark. Natural infection was associated with higher antibody responses, and IgG decline varied by age, sex, T-cell response, previous infection, and interval between vaccine doses.

IntroductionWe investigated humoral and T-cell responses within 12 months after first BNT162b2 vaccine in solid organ transplant (SOT) recipients and controls who had received at least three vaccine doses. Furthermore, we compared the immune response in participants with and without previous SARS-CoV-2 infection.MethodsWe included adult liver, lung, and kidney transplant recipients, and controls were selected from a parallel cohort of healthcare workers.ResultsAt 12th-month, the IgG geometric mean concentrations (GMCs) (P

Abstract Mechanisms keeping leukocytes distant of local inflammatory processes in a resting state despite systemic release of inflammatory triggers are a pivotal requirement for avoidance of overwhelming inflammation but are ill defined. Dimers of the alarmin S100A8/S100A9 activate Toll‐like receptor‐4 (TLR4) but extracellular calcium concentrations induce S100A8/S100A9‐tetramers preventing TLR4‐binding and limiting their inflammatory activity. So far, only antimicrobial functions of released S100A8/S100A9‐tetramers (calprotectin) are described. It is demonstrated that extracellular S100A8/S100A9 tetramers significantly dampen monocyte dynamics as adhesion, migration, and traction force generation in vitro and immigration of monocytes in a cutaneous granuloma model and inflammatory activity in a model of irritant contact dermatitis in vivo. Interestingly, these effects are not mediated by the well‐known binding of S100A8/S100A9‐dimers to TLR‐4 but specifically mediated by S100A8/S100A9‐tetramer interaction with CD69. Thus, the quaternary structure of these S100‐proteins determines distinct and even antagonistic effects mediated by different receptors. As S100A8/S100A9 are released primarily as dimers and subsequently associate to tetramers in the high extracellular calcium milieu, the same molecules promote inflammation locally (S100‐dimer/TLR4) but simultaneously protect the wider environment from overwhelming inflammation (S100‐tetramer/CD69).

Acute kidney injury (AKI) represents a common complication in critically ill patients that is associated with increased morbidity and mortality. In a murine AKI model induced by ischemia/reperfusion injury (IRI), we show that glutamine significantly decreases kidney damage and improves kidney function. We demonstrate that glutamine causes transcriptomic and proteomic reprogramming in murine renal tubular epithelial cells (TECs), resulting in decreased epithelial apoptosis, decreased neutrophil recruitment, and improved mitochondrial functionality and respiration provoked by an ameliorated oxidative phosphorylation. We identify the proteins glutamine gamma glutamyltransferase 2 (Tgm2) and apoptosis signal-regulating kinase (Ask1) as the major targets of glutamine in apoptotic signaling. Furthermore, the direct modulation of the Tgm2-HSP70 signalosome and reduced Ask1 activation resulted in decreased JNK activation, leading to diminished mitochondrial intrinsic apoptosis in TECs. Glutamine administration attenuated kidney damage in vivo during AKI and TEC viability in vitro under inflammatory or hypoxic conditions.

Abstract Background Excessive osteoclast activity, which is strongly stimulated by pro-inflammatory mediators, results in bone and cartilage degeneration as central features of many arthritides. Levels of the alarmin S100A8/A9 and interleukin (IL)-1β are both increased in arthritis patients and correlate with disease activity and progression of tissue erosion. We previously presented S100A8/A9 as a good biomarker for joint inflammation and arthritis pathology under circumstances of high IL-1 signaling in mice that lack the gene encoding IL-1 receptor antagonist (Il1rn −/− mice). Here, we investigated whether S100A8/A9 is also actively involved in the development of joint inflammation and both cartilage and bone pathology under these conditions by comparing Il1rn −/− mice with mice that have an additional deficiency for S100a9 (Il1rn −/− XS100a9 −/− ). Methods Il1rn −/− XS100a9 −/− on a BALB/c background were obtained by crossing S100a9 −/− mice and Il1rn −/− mice. Arthritis incidence and severity were macroscopically scored. Myeloid cell populations in the bone marrow and spleen were determined using flow cytometry. In vitro osteoclastogenesis of bone marrow cells was evaluated with TRAP staining. Microscopic joint inflammation, cartilage degeneration, and bone destruction were evaluated using histology of ankle joints of 12- and 20-week-old mice. Results Macroscopically scored arthritis severity was comparable between Il1rn −/− and Il1rn −/− XS100a9 −/− mice. Inflammation, cartilage erosion, and bone erosion were clearly present in 12-week-old mice of both strains lacking Il1rn −/− , but not significantly different between Il1rn −/− XS100a9 −/− and Il1rn −/− . Moreover, we observed that the numbers of neutrophils and monocytes were increased by the absence of Il1rn, which was affected by the absence of S100a9 only in the spleen but not in the bone marrow. In line with our other findings, the absence of S100a9 did not affect the osteoclastogenic potential of osteoclast precursors in the absence of Il1rn. Finally, in agreement with the findings in early arthritis development in 12-week-old mice, cartilage and bone erosion in 20-week-old mice was significantly higher in both Il1rn −/− strains, but the additional absence of S100a9 did not further affect tissue pathology. Conclusion S100A8/A9 deficiency does not significantly affect inflammation and joint destruction in mice with high IL1β signaling suggesting that S100A8/A9 is not essential for the development of arthritis under these conditions.

Abstract Background Acute tonsillitis represents one of the most frequent reasons patients seek primary medical care and otorhinolaryngology consultation. Therefore, recurrent episodes of acute tonsillitis (RAT), also called chronic tonsillitis, exhaust a substantial amount of medical and financial resources. Diagnosis of tonsillitis depends on a physical examination, which therefore does not allow for a reliable differentiation between viral and bacterial infection. However, the frequency of bacterial infections during the previous three years is currently being used as the major deciding factor in patient selection for tonsillectomy. The aim of the present study was to determine an objective biomarker to help in the identification of patients suffering from recurrent tonsillitis. Results By analyzing a panel of cytokines and chemokines in serum and saliva of patients with RAT compared to healthy controls, increased levels of IL-1ß (153.7 ± 48.5 pg/ml vs 23.3 ± 6.6 pg/ml, p = 0.021), IL-18 (120.2 ± 16.5 vs 50.6 ± 9.3 pg/ml, p = 0.007) and/or S100A8/A9 (996 ± 102 ng/ml vs 546 ± 86 ng/ml, p = 0.042) could be observed in patients suffering from RAT. Cut-off values of these parameters were determined and combined to a new RAT-score allowing for reliable identification of patients suffering from recurrent tonsillitis with a sensitivity of 95% and a specificity of 88%. Conclusion The RAT-score represents the first objective criterion as a tool for the diagnosis of recurrent tonsillitis and it also improves patient selection for tonsillectomy.

Abstract Background Physical activity (PA) patterns in children with juvenile idiopathic arthritis (JIA) over time are not well described. The aim of this study was to describe associations of physical activity (PA) with disease activity, function, pain, and psychosocial stress in the 2 years following diagnosis in an inception cohort of children with juvenile idiopathic arthritis (JIA). Methods In 82 children with newly diagnosed JIA, PA levels, prospectively determined at enrollment, 12 and 24 months using the Physical Activity Questionnaire for Children (PAQ-C) and Adolescents (PAQ-A) raw scores, were evaluated in relation to disease activity as reflected by arthritis activity (Juvenile Arthritis Disease Activity Score (JADAS-71)), function, pain, and psychosocial stresses using a linear mixed model approach. Results in the JIA cohort were compared to normative Pediatric Bone Mineral Accrual Study data derived from healthy children using z-scores. Results At enrollment, PA z-score levels of study participants were lower than those in the normative population (median z-score − 0.356; p = 0.005). At enrollment, PA raw scores were negatively associated with the psychosocial domain of the Juvenile Arthritis Quality of Life Questionnaire (r = − 0.251; p = 0.023). There was a significant decline in PAQ-C/A raw scores from baseline (median and IQR: 2.6, 1.4–3.1) to 24 months (median and IQR: 2.1, 1.4–2.7; p = 0.003). The linear mixed-effect model showed that PAQ-C/A raw scores in children with JIA decreased as age, disease duration, and ESR increased. The PAQ-C/A raw scores of the participants was also negatively influenced by an increase in disease activity as measured by the JADAS-71 (p

Purpose: As a promotor of tumor invasion and tumor microenvironment (TME) formation, the protein complex S100A8/S100A9 is associated with poor prognosis. Our aim was to further evaluate its origin and regulatory effects, and to establish an imaging biomarker for TME activity. Methods: S100A9−/−cells (ko) were created from syngeneic murine breast cancer 4T1 (high malignancy) and 67NR (low malignancy) wildtype (wt) cell lines and implanted into either female BALB/c wildtype or S100A9−/− mice (n = 10 each). Anti-S100A9-Cy5.5-targeted fluorescence reflectance imaging was performed at 0 h and 24 h after injection. Potential early changes of S100A9-presence under immune checkpoint inhibition (anti-PD-L1, n = 7 vs. rat IgG2b as isotype control, n = 3) were evaluated. Results: In S100A9−/−mice contrast-to-noise-ratios were significantly reduced for wt and S100A9−/−tumors. No significant differences were detected for 4T1 ko and 67NR ko cells as compared to wildtype cells. Under anti-PD-L1 treatment S100A9 presence significantly decreased compared with the control group. Conclusion: Our results confirm a secretion of S100A8/S100A9 by the TME, while tumor cells do not apparently release the protein. Under immune checkpoint inhibition S100A9-imaging reports an early decrease of TME activity. Therefore, S100A9-specific imaging may serve as an imaging biomarker for TME formation and activity.

Immune reactions are characterized by the rapid immigration of phagocytes into sites of inflammation. Meticulous regulation of these migratory processes is crucial for preventing uncontrolled and harmful phagocyte extravasation. S100A8/S100A9 is the major calcium-binding protein complex expressed in phagocytes. After release, this complex acts as a proinflammatory alarmin in the extracellular space, but the intracellular functions of these highly abundant proteins are less clear. Results of this study reveal an important role of S100A8/S100A9 in coordinated cytoskeleton rearrangement during migration. We found that S100A8/S100A9 was able to cross-link F-actin and microtubules in a calcium- and phosphorylation-dependent manner. Cells deficient in S100A8/S100A9 showed abnormalities in cell adhesion and motility. Missing cytoskeletal interactions of S100A8/S100A9 caused differences in the surface expression and activation of β1-integrins as well as in the regulation of Src/Syk kinase family members. Loss of S100A8/S100A9 led to dysregulated integrin-mediated adhesion and migration, resulting in an overall higher dynamic activity of non-activated S100A8/S100A9-deficient phagocytes. Our data suggest that intracellular S100A8/S100A9 is part of a novel regulatory mechanism that ensures the precise control necessary to facilitate the change between the quiescent and activated state of phagocytes.

Abstract S100A8/A9 (Calprotectin) serves as a biomarker for various inflammatory diseases, such as for peritonsillar abscess (PTA). Recently, the PTA score was developed for reliable PTA identification. It uses a combination of characteristic clinical symptoms and elevated calprotectin levels in serum and saliva to determine this score. Although well-established point-of-care tests (POCT) to determine serum or faecal calprotectin levels exist, a reliable and rapid tool to analyse salivary calprotectin has not yet been described. In this study, we analysed the potential of the QUANTUM BLUE sCAL Test (QBT, BÜHLMANN Laboratories AG, Switzerland) to determine S100A8/A9 levels during outpatient management. These QBT measurements are combined with other clinical factors to determine the PTA score. Significantly higher calprotectin levels were determined by QBT in patients with PTA compared to healthy controls. The receiver operating characteristic (ROC) curves for the QBT revealed cut-off values of 2940 ng/ml (sensitivity = 0.88, specificity = 0.78) in serum and 5310 ng/ml (sensitivity = 0.80, specificity = 0.50) in saliva. By adding the QBT results to determine PTA values, a ROC analysis provided a statistical cut-off score of 2.5 points to identify the existence of a PTA with a sensitivity of 100% and a specificity of 89.3%. The QUANTUM BLUE sCAL Test (QBT) is an appropriate POCT to determine serum and salivary calprotectin levels. Thus, PTA scores can be determined within a short time frame by applying the QBT during outpatient management.

The proinflammatory alarmins S100A8 and S100A9 are among the most abundant proteins in neutrophils and monocytes but are completely silenced after differentiation to macrophages. The molecular mechanisms of the extraordinarily dynamic transcriptional regulation of S100a8 and S100a9 genes, however, are only barely understood. Using an unbiased genome-wide CRISPR/Cas9 knockout (KO)-based screening approach in immortalized murine monocytes, we identified the transcription factor C/EBPδ as a central regulator of S100a8 and S100a9 expression. We showed that S100A8/A9 expression and thereby neutrophil recruitment and cytokine release were decreased in C/EBPδ KO mice in a mouse model of acute lung inflammation. S100a8 and S100a9 expression was further controlled by the C/EBPδ antagonists ATF3 and FBXW7. We confirmed the clinical relevance of this regulatory network in subpopulations of human monocytes in a clinical cohort of cardiovascular patients. Moreover, we identified specific C/EBPδ-binding sites within S100a8 and S100a9 promoter regions, and demonstrated that C/EBPδ-dependent JMJD3-mediated demethylation of H3K27me3 is indispensable for their expression. Overall, our work uncovered C/EBPδ as a novel regulator of S100a8 and S100a9 expression. Therefore, C/EBPδ represents a promising target for modulation of inflammatory conditions that are characterized by S100a8 and S100a9 overexpression.

Abstract Background Synovitis-associated pain is mediated by inflammatory factors that may include S100A8/9, which is able to stimulate nociceptive neurons via Toll-like receptor 4. In this study, we investigated the role of S100A9 in pain response during acute synovitis. Methods Acute synovitis was induced by streptococcal cell wall (SCW) injection in the knee joint of C57Bl/6 (WT) and S100A9 −/− mice. The expression of S100A8/A9 was determined in serum and synovium by ELISA and immunohistochemistry. Inflammation was investigated by 99mTc accumulation, synovial cytokine release, and histology at days 1, 2, and 7. To assess pain, weight distribution, gait analysis, and mechanical allodynia were monitored. Activation markers in afferent neurons were determined by qPCR and immunohistochemistry in the dorsal root ganglia (DRG). Differences between groups were tested using a one-way or two-way analysis of variance (ANOVA). Differences in histology were tested with a non-parametric Mann–Whitney U test. p values lower than 0.05 were considered significant. Results Intra-articular SCW injection resulted in increased synovial expression and serum levels of S100A8/A9 at day 1. These increased levels, however, did not contribute to the development of inflammation, since this was equal in S100A9 −/− mice. WT mice showed a significantly decreased percentage of weight bearing on the SCW hind paw on day 1, while S100A9 −/− mice showed no reduction. Gait analysis showed increased “limping” behavior in WT, but not S100A9 −/− mice. Mechanical allodynia was observed but not different between WT and S100A9 −/− when measuring paw withdrawal threshold. The gene expression of neuron activation markers NAV1.7, ATF3, and GAP43 in DRG was significantly increased in arthritic WT mice at day 1 but not in S100A9 −/− mice. Conclusions S100A8/9, released from the synovium upon inflammation, is an important mediator of pain response in the knee during the acute phase of inflammation.

Introduction Responses to COVID-19 vaccination in patients with chronic pulmonary diseases are poorly characterised. We aimed to describe humoral responses following two doses of BNT162b2 mRNA COVID-19 vaccine and identify risk factors for impaired responses.Methods Prospective cohort study including adults with chronic pulmonary diseases and healthcare personnel as controls (1:1). Blood was sampled at inclusion, 3 weeks, 2 and 6 months after first vaccination. We reported antibody concentrations as geometric means with 95% CI of receptor binding domain (RBD)-IgG and neutralising antibody index of inhibition of ACE-2/RBD interaction (%). A low responder was defined as neutralising index in the lowest quartile (primary outcome) or RBD-IgG

BackgroundPrevious studies have indicated inferior responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination in solid organ transplant (SOT) recipients. We examined the development of anti-receptor-binding domain (RBD) immunoglobulin G (IgG) after two doses of BNT162b2b in SOT recipients 6 months after vaccination and compared to that of immunocompetent controls.MethodsWe measured anti-RBD IgG after two doses of BNT162b2 in 200 SOT recipients and 200 matched healthy controls up to 6 months after first vaccination. Anti-RBD IgG concentration and neutralizing capacity of antibodies were measured at first and second doses of BNT162b2 and 2 and 6 months after the first dose. T-cell responses were measured 6 months after the first dose.ResultsIn SOT recipients, geometric mean concentration (GMC) of anti-RBD IgG increased from first to second dose (1.14 AU/ml, 95% CI 1.08–1.24 to 11.97 AU/ml, 95% CI 7.73–18.77) and from second dose to 2 months (249.29 AU/ml, 95% CI 153.70–385.19). Six months after the first vaccine, anti-RBD IgG declined (55.85 AU/ml, 95% CI 36.95–83.33). At all time points, anti-RBD IgG was lower in SOT recipients than that in controls. Fewer SOT recipients than controls had a cellular response (13.1% vs. 59.4%, p < 0.001). Risk factors associated with humoral non-response included age [relative risk (RR) 1.23 per 10-year increase, 95% CI 1.11–1.35, p < 0.001], being within 1 year from transplantation (RR 1.55, 95% CI 1.30–1.85, p < 0.001), treatment with mycophenolate (RR 1.54, 95% CI 1.09–2.18, p = 0.015), treatment with corticosteroids (RR 1.45, 95% CI 1.10–1.90, p = 0.009), kidney transplantation (RR 1.70, 95% CI 1.25–2.30, p = 0.001), lung transplantation (RR 1.63, 95% CI 1.16–2.29, p = 0.005), and de novo non-skin cancer comorbidity (RR 1.52, 95% CI, 1.26–1.82, p < 0.001).ConclusionImmune responses to BNT162b2 are inferior in SOT recipients compared to healthy controls, and studies aiming to determine the clinical impact of inferior vaccine responses are warranted.

Background: It is widely accepted that SARS-CoV-2 causes a dysregulation of immune and coagulation processes. In severely affected patients, viral sepsis may result in life endangering multiple organ dysfunction. Furthermore, most therapies for COVID-19 patients target either the immune system or coagulation processes. As the exact mechanism causing SARS-CoV-2-induced morbidity and mortality was unknown, we started an in-depth analysis of immunologic and coagulation processes. Methods: 127 COVID-19 patients were treated at the University Hospital Essen, Germany, between May 2020 and February 2022. Patients were divided according to their maximum COVID-19 WHO ordinal severity score (WHO 0–10) into hospitalized patients with a non-severe course of disease (WHO 4–5, n = 52) and those with a severe course of disease (WHO 6–10, n = 75). Non-infected individuals served as healthy controls (WHO 0, n = 42). Blood was analyzed with respect to cell numbers, clotting factors, as well as pro- and anti-inflammatory mediators in plasma. As functional parameters, phagocytosis and inflammatory responses to LPS and antigen-specific stimulation were determined in monocytes, granulocytes, and T cells using flow cytometry. Findings: In the present study, immune and coagulation systems were analyzed simultaneously. Interestingly, many severe COVID-19 patients showed an upregulation of pro-inflammatory mediators and at the same time clear signs of immunosuppression. Furthermore, severe COVID-19 patients not only exhibited a disturbed immune system, but in addition showed a pronounced pro-coagulation phenotype with impaired fibrinolysis. Therefore, our study adds another puzzle piece to the already complex picture of COVID-19 pathology implying that therapies in COVID-19 must be individualized. Conclusion: Despite years of research, COVID-19 has not been understood completely and still no therapies exist, fitting all requirements and phases of COVID-19 disease. This observation is highly reminiscent to sepsis. Research in sepsis has been going on for decades, while the disease is still not completely understood and therapies fitting all patients are lacking as well. In both septic and COVID-19 patients, immune activation can be accompanied by immune paralysis, complicating therapeutic intervention. Accordingly, therapies that lower immune activation may cause detrimental effects in patients, who are immune paralyzed by viral infections or sepsis. We therefore suggest individualizing therapies and to broaden the spectrum of immunological parameters analyzed before therapy. Only if the immune status of a patient is understood, can a therapeutic intervention be successful.

Abstract Objective To investigate whether calprotectin (S100A8/A9 or MRP8/14), an inflammatory complex released by monocytes, could indicate residual subclinical inflammation in rheumatoid arthritis (RA) patients who are in stable remission on disease-modifying anti-rheumatic drugs (DMARDs) and serve as a marker for disease flare after DMARD tapering. Methods We used data from two trials. Patients from the IMPROVED study had early (

Sepsis is defined by life-threatening organ dysfunction mediated by the host’s response to infection. This can result in septic dyslipidemia, which is involved in the neutralization of pathogen-related lipids. Knowledge of the regulatory mechanisms of septic dyslipidemia is incomplete. The cytokine betatrophin/Angiopoietin-like protein 8 (ANGPTL8) plays a role in the regulation of triacylglyceride metabolism, though its function in septic dyslipidemia remains unknown. Sixty-six patients were enrolled in a cross-sectional study. Circulating concentrations and adipose tissue (AT) mRNA expression of betatrophin/ANGPTL8 were studied in patients suffering from peritoneal sepsis. Insulin-resistant individuals and subjects without metabolic derangement/systemic inflammation were enrolled as controls. All underwent open abdominal surgery. Circulating betatrophin/ANGPTL8 was analyzed by an enzyme-linked immunosorbent assay and AT mRNA expression levels were assessed by real-time PCR. Standard laboratory analyses including lipid electrophoresis were evaluated. Sepsis patients showed pronounced septic dyslipidemia (p < 0.05 for all major lipid classes). Despite comparable betatrophin/ANGPTL8 mRNA expression in AT (p = 0.24), we found significantly increased circulating betatrophin/ANGPTL8 with septic dyslipidemia (p = 0.009). Expression levels of betatrophin/ANGPTL8 in AT correlated with circulating concentrations in both control groups (r = 0.61; p = 0.008 and r = 0.43; p = 0.034), while this association was undetectable in sepsis. After stratification, betatrophin/ANGPTL8 remained associated with hypertriacylglyceridemia (p < 0.05).

Acute respiratory distress syndrome (ARDS) due to pulmonary infections is associated with high morbidity and mortality. Upon inflammation, the alarmin S100A8/A9 is released and stimulates neutrophil recruitment mainly via binding to Toll-like receptor 4 (TLR4). TLR4 is also expressed on platelets, which modulate the immune response through direct interaction with leukocytes. In a murine model of Klebsiella pneumoniae-induced pulmonary inflammation, global S100A9 deficiency resulted in diminished neutrophil recruitment into the lung alveoli and neutrophil accumulation in the intravascular space, indicating an impaired neutrophil migration. A lack of TLR4 on platelets resulted in reduced neutrophil counts in the whole lung, emphasising the impact of TLR4-mediated platelet activity on neutrophil behaviour. Flow cytometry-based analysis indicated elevated numbers of platelet–neutrophil complexes in the blood of S100A9−/− mice. Intravital microscopy of the murine cremaster muscle confirmed these findings and further indicated a significant increase in neutrophil–platelet complex formation in S100A9−/− mice, which was reversed by administration of the S100A8/A9 tetramer. An in vitro bilayer assay simulated the murine alveolar capillary barrier during inflammation and validated significant differences in transmigration behaviour between wild-type and S100A9−/− neutrophils. This study demonstrates the role of S100A8/A9 during platelet–neutrophil interactions and neutrophil recruitment during pulmonary inflammation.

Objective To develop an ultrasonographic image acquisition protocol and a joint-specific scoring system for synovitis with reference atlas in patients with juvenile idiopathic arthritis (JIA) and to assess the reliability of the system.Methods Seven rheumatologists with extensive ultrasound experience developed a scanning protocol and a semiquantitative joint-specific scoring system for B-mode (BM) synovitis for the elbow, wrist, metacarpophalangeal 2–3, proximal interphalangeal 2–3, hip, knee, ankle and metatarsophalangeal 2–3 joints. An ultrasonographic reference atlas for BM synovitis, divided in four age groups (2–4, 5–8, 9–12, 13–18 years), and power Doppler (PD) activity was then developed. Reliability was assessed for all joints on still images and in a live exercise including 10 patients with JIA, calculated by intraclass correlation coefficient (ICC) and weighted kappa.Results A scanning protocol and scoring system for multiple joints with reference atlas composed of images with four different score levels for BM and PD were developed. Still image scoring for BM synovitis on joint level showed good to excellent intra-reader reliability (ICC/kappa ranges: 0.75–0.95/0.63–0.91) and moderate to excellent inter-reader reliability (ICC/kappa ranges: 0.89–0.99/0.50–0.91). Still image scoring for PD activity showed excellent intra-reader and inter-reader reliability (ICC/kappa: 0.96/0.91 and ICC/kappa: 0.97/0.80, respectively). In the live scoring, inter-reader reliability (ICC/kappa) was moderate to excellent for BM synovitis (0.94/0.51) and PD activity (0.91/0.60).Conclusion An ultrasonographic image acquisition protocol and joint-specific scoring system with reference atlas were developed and demonstrated moderate to excellent reliability for scoring of synovitis in patients with JIA. This can be a valuable tool in clinical practice and future research.

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Neonatal immune responses are known to differ to those of an adult immune response. Here Dreschers and colleagues, compare macrophage populations derived from cord blood or adult peripheral blood and show a metabolic impairment of glycolysis in macrophages derived from cord blood which may impair response to infective scenarios such as sepsis.

Abstract Background Measurement of gylcated hemoglobin A1c (HbA1c) plays a central role in monitoring quality of antidiabetic therapy and in the diagnosis of diabetes. Several studies report increased levels of HbA1c in nondiabetic elderly. However, this observation did not reach incorporation into daily clinical practice or the respective guidelines. The present study aimed to evaluate HbA1c levels in relation to age in two independent population-based cohorts and to derive age-specific reference intervals. Methods Four thousand two hundred sixty three participants from the Study of Health in Pomerania (SHIP-0) and 4402 participants from the independent study SHIP-Trend were included. HbA1c was determined by means of high-performance liquid chromatography. Multivariable linear regression models were performed. Reference intervals for HbA1c were determined. Results Reference intervals were derived from a healthy subpopulation with the upper reference limit (URL) for HbA1c of 42.1 mmol/Mol (6.0%) for individuals aged 20–39 years increasing to 43.2 mmol/Mol (6.1%) for individuals aged 40–59 years. For people aged ≥60 years the URL was 47.5 mmol/Mol (6.5%). In both study populations an increase in HbA1c with age was observed. ANOVA revealed up to 8.5 mmol/Mol (0.77%) or 7.3 mmol/Mol (0.68%) higher estimated mean levels of HbA1c in the oldest compared to the youngest age group in SHIP-0 or SHIP-trend, respectively. Linear regression analyses confirmed the positive associations of HbA1c with age which was independent of BMI Conclusion The present study confirmed the previously observed increase of HbA1c with increasing age in non-diabetic individuals. As a consequence age-dependent reference values for HbA1c were derived from two large and well defined reference populations. Implementation of them into daily practice may improve patient care and diagnosis of diabetes and reduce the risk of misdiagnosis and subsequent overtreatment of diabetes in elderly patients.

Monocytes and macrophages are central players of the innate immune response and play a pivotal role in the regulation of inflammation. Thereby, they actively participate in all phases of the immune response, from initiating inflammation and triggering the adaptive immune response, through to the clearance of cell debris and resolution of inflammation. In this review, we described the mechanisms of monocyte and macrophage adaptation to rapidly changing microenvironmental conditions and discussed different forms of macrophage polarization depending on the environmental cues or pathophysiological condition. Therefore, special focus was placed on the tight regulation of the pro- and anti-inflammatory immune response, and the diverse functions of S100A8/S100A9 proteins and the scavenger receptor CD163 were highlighted, respectively. We paid special attention to the function of pro- and anti-inflammatory macrophages under pathological conditions.

Psoriasis is one of the most common immune-mediated inflammatory skin diseases. Expression and secretion of two pro-inflammatory molecules of the S100-alarmin family, S100A8 and S100A9, in keratinocytes is a hallmark of psoriasis, which is also characterized by an altered differentiation of keratinocytes. Dimers of S100A8/S100A9 (calprotectin) bind to Toll-like receptor 4 and induce an inflammatory response in target cells. Targeted deletion of S100A9 reduced the inflammatory phenotype of psoriasis-like inflammation in mice. A role of S100-alarmins in differentiation and activation of keratinocytes was suggested but has been never shown in primary keratinocytes. We now confirm that induction of S100-alarmins in an imiquimod-induced murine model of psoriasis-like skin inflammation was associated with an increased expression of interleukin (IL)-1α, IL-6, IL-17A, or TNFα. This association was confirmed in transcriptome data obtained from controls, lesional and non-lesional skin of psoriasis patients, and a down-regulation of S100-alarmin expression after IL-17 directed therapy. However, analyzing primary S100A9−/− keratinocytes we found that expression of S100A8/S100A9 has no significant role for the maturation and inflammatory response pattern of keratinocytes. Moreover, keratinocytes are no target cells for the pro-inflammatory effects of S100A8/S100A9. However, different cytokines, especially IL-17A and F, highly abundant in psoriasis, strongly induced expression of S100-alarmins preferentially during early maturation stages of keratinocytes. Our data indicate that expression of S100A8 and S100A9 does not primarily influence maturation or activation of keratinocytes but rather represents the inflammatory response of these cells during psoriasis.

Intra-abdominal infection (peritonitis) is a leading cause of severe disease in surgical intensive care units, as over 70% of patients diagnosed with peritonitis develop septic shock. A critical role of the immune system is to return to homeostasis after combating infection. S100A8/A9 (calprotectin) is an antimicrobial and pro-inflammatory protein complex used as a biomarker for diagnosis of numerous inflammatory disorders. Here we describe the role of S100A8/A9 in inflammatory collateral tissue damage (ICTD). Using a mouse model of disseminated intra-abdominal candidiasis (IAC) in wild-type and S100A8/A9-deficient mice in the presence or absence of S100A9 inhibitor paquinimod, the role of S100A8/A9 during ICTD and fungal clearance were investigated. S100A8/A9-deficient mice developed less ICTD than wild-type mice. Restoration of S100A8/A9 in knockout mice by injection of recombinant protein resulted in increased ICTD and fungal clearance comparable to wild-type levels. Treatment with paquinimod abolished ICTD and S100A9-deficient mice showed increased survival compared to wild-type littermates. The data indicates that S100A8/A9 controls ICTD levels and antimicrobial activity during IAC and that targeting of S100A8/A9 could serve as promising adjunct therapy against this challenging disease.

Monocyte differentiation to macrophages is triggered by migration across the endothelial barrier, which is constituted by both endothelial cells and their underlying basement membrane. We address here the role of the endothelial basement membrane laminins (laminins 411 and 511) in this monocyte to macrophage switch. Chimeric mice carrying CX3CR1-GFP bone marrow were employed to track CCL2-induced monocyte extravasation in a cremaster muscle model using intravital microscopy, revealing faster extravasation in mice lacking endothelial laminin 511 (Tek-cre::Lama5−/−) and slower extravasation in mice lacking laminin 411 (Lama4−/−). CX3CR1-GFPlow extravasating monocytes were found to have a higher motility at laminin 511 low sites and to preferentially exit vessels at these sites. However, in vitro experiments reveal that this is not due to effects of laminin 511 on monocyte migration mode nor on the tightness of the endothelial barrier. Rather, using an intestinal macrophage replenishment model and in vitro differentiation studies, we demonstrate that laminin 511, together with the attached endothelium, promote monocyte differentiation to macrophages. Macrophage differentiation is associated with a change in integrin profile, permitting differentiating macrophages to distinguish between laminin 511 high and low areas and to preferentially migrate across laminin 511 low sites. These studies highlight the endothelial basement membrane as a critical site for monocyte differentiation to macrophages, which may be relevant to the differentiation of other cells at vascular niches.

Abstract Background Musculoskeletal Ultrasonography (MSUS) is an important tool for the clinical assessment in Juvenile Idiopathic Arthritis (JIA). The objective of this study was to evaluate the reliability of MSUS to detect elementary lesions: synovitis, tenosynovitis, cartilage damage and bone erosions in the wrist and metacarpal (MCP) joints of patients with JIA. Methods Thirty children in various subgroups of JIA according to ILAR criteria, were included in this cross-sectional study. Clinical data including painful, swollen and limited joints were recorded. Five rheumatologist ultrasonographers, blinded to the clinical evaluation, evaluated the presence of elementary lesions in the wrist and MCP 2 and 3 joints bilaterally. The synovitis was graded in B-Mode and Power Doppler (PD). In addition to descriptive statistics intra- and inter-observer reliability was calculated using Cohen’s kappa according to Landis and Koch. Results US detected more synovitis than the clinical examination (62% vs 28%, 30% vs 23% and 22% vs 17% in the wrist, second and third MCP joints respectively). The intra-observer concordance for synovitis in all joints was excellent in B-Mode (k 0.84 .63–1.0 p = 0.001), except for MCP 2, where it was good (0.61, IC 95% .34–89, p = 0.001). For both modalities (PD, B-Mode) tenosynovitis, cartilage damage and bone erosions it was also excellent. Regarding synovitis grading the concordance was excellent for all grades (0.83–1.0, IC 95% 0.51.1.0, p = 0.001), except for grade 1 where it was good (0.61, IC 95% 0.43–.83, p = 0.001). Reliability inter-observer for grayscale synovitis (0.67–0.95, IC 95% 0.67–1.0, p = 0.001), tenosynovitis grayscale (0.89, IC 95% 0.78–0.99, p.001), damage cartilage (0.89, IC 95% 0.78–0.99, p = 0.001), PD (0.66, IC 95% 0.39–1.0, p = 0.001). The concordance for grading synovitis was excellent, but for grayscale grade 1 and 2 (.66, IC 95% .53–.74, p = 0.007) and PD grade 1 and 2 (0.63, IC 95% .58–.91, p = 004) was good. Conclusions The intra- and inter-observer reliability of MSUS for inflammatory and structural lesions is good to excellent for the wrist and MCP in patients with JIA.

Abstract Background Osteoclast-mediated bone erosion is a central feature of rheumatoid arthritis (RA). Immune complexes, present in a large percentage of patients, bind to Fcγ receptors (FcγRs), thereby modulating the activity of immune cells. In this study, we investigated the contribution of FcγRs, and FcγRIV in particular, during antigen-induced arthritis (AIA). Methods AIA was induced in knee joints of wild-type (WT), FcγRI,II,III−/−, and FcγRI,II,III,IV−/− mice. Bone destruction, numbers of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts, and inflammation were evaluated using histology; expression of the macrophage marker F4/80, neutrophil marker NIMPR14, and alarmin S100A8 was evaluated using immunohistochemistry. The percentage of osteoclast precursors in the bone marrow was determined using flow cytometry. In vitro osteoclastogenesis was evaluated with TRAP staining, and gene expression was assessed using real-time PCR. Results FcγRI,II,III,IV−/− mice showed decreased bone erosion compared with WT mice during AIA, whereas both the humoral and cellular immune responses against methylated bovine serum albumin were not impaired in FcγRI,II,III,IV−/− mice. The percentage of osteoclast precursors in the bone marrow of arthritic mice and their ability to differentiate into osteoclasts in vitro were comparable between FcγRI,II,III,IV−/− and WT mice. In line with these observations, numbers of TRAP+ osteoclasts on the bone surface during AIA were comparable between the two groups. Inflammation, a process that strongly activates osteoclast activity, was reduced in FcγRI,II,III,IV−/− mice, and of note, mainly decreased numbers of neutrophils were present in the joint. In contrast to FcγRI,II,III,IV−/− mice, AIA induction in knee joints of FcγRI,II,III−/− mice resulted in increased bone erosion, inflammation, and numbers of neutrophils, suggesting a crucial role for FcγRIV in the joint pathology by the recruitment of neutrophils. Finally, significant correlations were found between bone erosion and the number of neutrophils present in the joint as well as between bone erosion and the number of S100A8-positive cells, with S100A8 being an alarmin strongly produced by neutrophils that stimulates osteoclast resorbing activity. Conclusions FcγRs play a crucial role in the development of bone erosion during AIA by inducing inflammation. In particular, FcγRIV mediates bone erosion in AIA by inducing the influx of S100A8/A9-producing neutrophils into the arthritic joint.

Sedentary behavior constitutes a pandemic health threat contributing to the pathophysiology of obesity and type 2 diabetes (T2D). Sedentarism is further associated with liver disease and particularly with nonalcoholic/metabolic dysfunction associated fatty liver disease (NAFLD/MAFLD). Insulin resistance (IR) represents an early pathophysiologic key element of NAFLD/MAFLD, prediabetes and T2D. Current treatment guidelines recommend regular physical activity. There is evidence, that physical exercise has impact on a variety of molecular pathways, such as AMP-activated protein kinase and insulin signaling as well as glucose transporter 4 translocation, modulating insulin action, cellular substrate flow and in particular ectopic lipid and glycogen storage in a positive manner. Therefore, physical exercise can lead to substantial clinical benefit in persons with diabetes and/or NAFLD/MAFLD. However, experience from long term observational studies shows that the patients’ motivation to exercise regularly appears to be a major limitation. Strategies to integrate everyday physical activity (i.e., nonexercise activity thermogenesis) in lifestyle treatment schedules might be a promising approach. This review aggregates evidence on the impact of regular physical activity on selected molecular mechanisms as well as clinical outcomes of patients suffering from IR and NAFLD/MAFLD.

Osteoarthritis (OA) is a destructive disease of the joint with age and obesity being its most important risk factors. Around 50% of OA patients suffer from inflammation of the synovial joint capsule, which is characterized by increased abundance and activation of synovial macrophages that produce reactive oxygen species (ROS) via NADPH-oxidase 2 (NOX2). Both ROS and high blood levels of low-density lipoprotein (LDL) are implicated in OA pathophysiology, which may interact to form oxidized LDL (oxLDL) and thereby promote disease. Therefore, targeting NOX2 could be a viable treatment strategy for OA. Collagenase-induced OA (CiOA) was used to compare pathology between wild-type (WT) and Nox2 knockout (Nox2−/−) C57Bl/6 mice. Mice were either fed a standard diet or Western diet (WD) to study a possible interaction between NOX2-derived ROS and LDL. Synovial inflammation, cartilage damage and ectopic bone size were assessed on histology. Extracellular ROS production by macrophages was measured in vitro using the Amplex Red assay. Nox2−/− macrophages produced basal levels of ROS but were unable to increase ROS production in response to the alarmin S100A8 or the phorbol ester PMA. Interestingly, Nox2 deficiency reduced cartilage damage, synovial lining thickness and ectopic bone size, whereas these disease parameters were not affected by WD-feeding. These results suggest that NOX2-derived ROS are involved in CiOA development.

Abstract Background Monocytes are dominant cells present within the inflamed synovium during osteoarthritis (OA). In mice, two functionally distinct monocyte subsets are described: pro-inflammatory Ly6Chigh and patrolling Ly6Clow monocytes. Alarmins S100A8/A9 locally released by the synovium during inflammatory OA for prolonged periods may be dominant proteins involved in stimulating recruitment of Ly6Chigh monocytes from the circulation to the joint. Our objective was to investigate the role of S100A8/A9 in the mobilization of Ly6Chigh and Ly6Clow monocytic populations to the inflamed joint in collagenase-induced OA (CiOA). Method S100A8 was injected intra-articularly to investigate monocyte influx. CiOA was induced by injection of collagenase into knee joints of wild-type C57BL/6 (WT), and S100a9-/- mice. Mice were sacrificed together with age-matched saline-injected control mice (n = 6/group), and expression of monocyte markers, pro-inflammatory cytokines, and chemokines was determined in the synovium using ELISA and RT-qPCR. Cells were isolated from the bone marrow (BM), spleen, blood, and synovium and monocytes were identified using FACS. Results S100A8/A9 was highly expressed during CiOA. Intra-articular injection of S100A8 leads to elevated expression of monocyte markers and the monocyte-attracting chemokines CCL2 and CX3CL1 in the synovium. At day 7 (d7) after CiOA induction in WT mice, numbers of Ly6Chigh, but not Ly6Clow monocytes, were strongly increased (7.6-fold) in the synovium compared to saline-injected controls. This coincided with strong upregulation of CCL2, which preferentially attracts Ly6Chigh monocytes. In contrast, S100a9-/- mice showed a significant increase in Ly6Clow monocytes (twofold) within the synovium at CiOA d7, whereas the number of Ly6Chigh monocytes remained unaffected. In agreement with this finding, the Ly6Clow mobilization marker CX3CL1 was significantly higher within the synovium of S100a9-/- mice. Next, we studied the effect of S100A8/A9 on release of Ly6Chigh monocytes from the BM into the circulation. A 14% decrease in myeloid cells was found in WT BM at CiOA d7. No decrease in myeloid cells in S100a9-/- BM was found, suggesting that S100A8/A9 promotes the release of myeloid populations from the BM. Conclusion Induction of OA locally leads to strongly elevated S100A8/A9 expression and an elevated influx of Ly6Chigh monocytes from the BM to the synovium.

Abstract Eosinophils are bone marrow-derived cells that have been largely implicated in Th2-associated diseases. Recent data highlights a key role for eosinophils in mucosal innate immune responses especially in the gastrointestinal (GI) tract, which is one of the largest eosinophil reservoirs in the body. Although eosinophils express and synthesize a plethora of proteins that can mediate their effector activities, the transcriptome signature of eosinophils in mucosal inflammation and subsequent repair has been considerably overlooked. We demonstrate that eosinophils are recruited to the colon in acute inflammatory stages where they promote intestinal inflammation and remain in substantial numbers throughout the mucosal healing process. Microarray analysis of primary colonic eosinophils that were sorted at distinct stages of mucosal inflammation and repair revealed dynamic regulation of colonic eosinophil mRNA expression. The clinically relevant genes s100a8 and s100a9 were strikingly increased in colonic eosinophils (up to 550-fold and 80-fold, respectively). Furthermore, local and systemic expression of s100a8 and s100a9 were nearly diminished in eosinophil-deficient ΔdblGATA mice, and were re-constituted upon adoptive transfer of eosinophils. Taken together, these data may provide new insight into the involvement of eosinophils in colonic inflammation and repair, which may have diagnostic and therapeutic implications.

The early inflammatory skin micromilieu affects resistance in experimental infection with Leishmania major. We pursue the concept that macrophages, which take up parasites during early infection, exert decisive influence on the inflammatory micromilieu after infection. In order to analyze their distinctive potential, we identified differentially regulated genes of murine granuloma macrophages (GMΦ) from resistant and susceptible mice after their infection with metacyclic Leishmania major. We found induction of several cytokines in GMΦ from both strains and a stronger upregulation of the transcription factor aryl hydrocarbon receptor (AhR) in GMΦ from resistant mice. Using both an AhR agonist and antagonist we demonstrated that AhR is involved in Leishmania-induced production of TNF in macrophages. In vivo, single local injection of an AhR agonist in early lesions of susceptible mice caused an increased induction of Tnf and other cytokines in the skin. Importantly, local agonist treatment led to a reduction of disease severity, reduced parasite loads and a weaker Th2 response. Our results demonstrate that local activation of AhR has a beneficial effect in experimental leishmaniasis.

Uncontrolled inflammation is a leading cause of many clinically relevant diseases. Current therapeutic strategies focus mainly on immunosuppression rather than on the mechanisms of inflammatory resolution. Glucocorticoids (GCs) are still the most widely used anti-inflammatory drugs. GCs affect most immune cells but there is growing evidence for cell type specific mechanisms. Different subtypes of monocytes and macrophages play a pivotal role both in generation as well as resolution of inflammation. Activation of these cells by microbial products or endogenous danger signals results in production of pro-inflammatory mediators and initiation of an inflammatory response. GCs efficiently inhibit these processes by down-regulating pro-inflammatory mediators from macrophages and monocytes. On the other hand, GCs act on “naïve” monocytes and macrophages and induce anti-inflammatory mediators and differentiation of anti-inflammatory phenotypes. GC-induced anti-inflammatory monocytes have an increased ability to migrate toward inflammatory stimuli. They remove endo- and exogenous danger signals by an increased phagocytic capacity, produce anti-inflammatory mediators and limit T-cell activation. Thus, GCs limit amplification of inflammation by repressing pro-inflammatory macrophage activation and additionally induce anti-inflammatory monocyte and macrophage populations actively promoting resolution of inflammation. Further investigation of these mechanisms should lead to the development of novel therapeutic strategies to modulate undesirable inflammation with fewer side effects via induction of inflammatory resolution rather than non-specific immunosuppression.