Acute Disruption of Bone Marrow B Lymphopoiesis and Apoptosis of Transitional and Marginal Zone B Cells in the Spleen following a Blood-Stage Plasmodium chabaudi Infection in Mice

B cells and antibodies are essential for the protective immune response against a blood-stage Plasmodium infection. Although extensive research has focused on memory as well as plasma B-cell responses during infection, little is known about how malaria affects B-cell development and splenic maturation into marginal zone B (MZB) and follicular B (FoB) cells. In this study, we show that acute Plasmodium chabaudi AS infection in C57Bl/6 mice causes severe disruption of B lymphopoiesis in the bone marrow, affecting in particular pro-, pre-, and immature B cells as well as the expression of the bone marrow B-cell retention chemokine CXCL12. In addition, elevated apoptosis of transitional T2 and marginal zone (MZ) B cells was observed during and subsequent to the control of the first wave of parasitemia. In contrast, Folllicular (Fo) B cells levels were retained in the spleen throughout the infection, suggesting that these are essential for parasite clearance and proper infection control

Insufficiently Defined Genetic Background Confounds Phenotypes in Transgenic Studies As Exemplified by Malaria Infection in Tlr9 Knockout Mice

The use of genetically modified mice, i.e. transgenic as well as gene knockout (KO) and knock-in mice, has become an established tool to study gene function in many animal models for human diseases . However, a gene functions in a particular genomic context. This implies the importance of a well-defined homogenous genetic background for the analysis and interpretation of phenotypes associated with genetic mutations. By studying a Plasmodium chabaudi chabaudi AS (PcAS) malaria infection in mice bearing a TLR9 null mutation, we found an increased susceptibility to infection, i.e. higher parasitemia levels and increased mortality. However, this was not triggered by the deficient TLR9 gene itself. Instead, this disease phenotype was dependent on the heterogeneous genetic background of the mice, which appeared insufficiently defined as determined by single nucleotide polymorphism (SNP) analysis. Hence, it is of critical importance to study gene KO phenotypes on a homogenous genetic background identical to that of their wild type (WT) control counterparts. In particular, to avoid problems related to an insufficiently defined genetic background, we advocate that for each study involving genetically modified mice, at least a detailed description of the origin and genetic background of both the WT control and the altered strain of mice is essential

Mast cells protect from post-traumatic spinal cord damage in mice by degrading inflammation-associated cytokines via mouse mast cell protease 4

AbstractMast cells (MCs) are found abundantly in the central nervous system and play a complex role in neuroinflammatory diseases such as multiple sclerosis and stroke. In the present study, we show that MC-deficient KitW-sh/W-sh mice display significantly increased astrogliosis and T cell infiltration as well as significantly reduced functional recovery after spinal cord injury compared to wildtype mice. In addition, MC-deficient mice show significantly increased levels of MCP-1, TNF-α, IL-10 and IL-13 protein levels in the spinal cord. Mice deficient in mouse mast cell protease 4 (mMCP4), an MC-specific chymase, also showed increased MCP-1, IL-6 and IL-13 protein levels in spinal cord samples and a decreased functional outcome after spinal cord injury. A degradation assay using supernatant from MCs derived from either mMCP4−/− mice or controls revealed that mMCP4 cleaves MCP-1, IL-6, and IL-13 suggesting a protective role for MC proteases in neuroinflammation. These data show for the first time that MCs may be protective after spinal cord injury and that they may reduce CNS damage by degrading inflammation-associated cytokines via the MC-specific chymase mMCP4

Hepatocyte MyD88 affects bile acids, gut microbiota and metabolome contributing to regulate glucose and lipid metabolism

OBJECTIVE: To examine the role of hepatocyte myeloid differentiation primary-response gene 88 (MyD88) on glucose and lipid metabolism. DESIGN: To study the impact of the innate immune system at the level of the hepatocyte and metabolism, we generated mice harbouring hepatocyte-specific deletion of MyD88. We investigated the impact of the deletion on metabolism by feeding mice with a normal control diet or a high-fat diet for 8 weeks. We evaluated body weight, fat mass gain (using time-domain nuclear magnetic resonance), glucose metabolism and energy homeostasis (using metabolic chambers). We performed microarrays and quantitative PCRs in the liver. In addition, we investigated the gut microbiota composition, bile acid profile and both liver and plasma metabolome. We analysed the expression pattern of genes in the liver of obese humans developing non-alcoholic steatohepatitis (NASH). RESULTS: Hepatocyte-specific deletion of MyD88 predisposes to glucose intolerance, inflammation and hepatic insulin resistance independently of body weight and adiposity. These phenotypic differences were partially attributed to differences in gene expression, transcriptional factor activity (ie, peroxisome proliferator activator receptor-α, farnesoid X receptor (FXR), liver X receptors and STAT3) and bile acid profiles involved in glucose, lipid metabolism and inflammation. In addition to these alterations, the genetic deletion of MyD88 in hepatocytes changes the gut microbiota composition and their metabolomes, resembling those observed during diet-induced obesity. Finally, obese humans with NASH displayed a decreased expression of different cytochromes P450 involved in bioactive lipid synthesis. CONCLUSIONS: Our study identifies a new link between innate immunity and hepatic synthesis of bile acids and bioactive lipids. This dialogue appears to be involved in the susceptibility to alterations associated with obesity such as type 2 diabetes and NASH, both in mice and humans

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Interplay of hemozoin and host factors in malaria immunopathogenesis

Malaria is a widespread mosquito-borne infectious disease affecting about 500 million people yearly and resulting in over 1 million of deaths, particularly in sub-Saharan Africa. The majority of malaria-related mortalities is caused by infections of Plasmodium falciparum and occurs mostly in young infants under the age of five years. Also, primigravidae are at increased risk of pregnancy-associated malaria. Although people living in endemic regions do acquire clinical immunity when frequently infected, no effective malaria vaccine is yet available. An inadequate understanding of the immune mechanisms leading to the development of natural immunity is a critical factor contributing to this failure. To date, RTS,S is the most clinically advanced subunit candidate-vaccine in the development pipeline and provides &plusmn; 50% protection.Upon the bite of an infected mosquito,sporozoites enter the bloodstream and migrate to the liver. After passing through a single phase of asymptomatic schizogony in the hepatocytes, merozoites are released in the blood circulation. This asexual blood-stage parasite infects the red blood cell (RBC) and starts a cycle of erythrocytic schizogony, accompanied by the induction of malaria disease symptoms ranging from fever to life-threatening pathology, e.g. severe malaria anemia and cerebral malaria. Many of the severe malaria-associated pathologies have an immunological basis, as an inappropriately regulated proinflammatory immune response elicited by parasitized RBCs and high doses of parasite toxins significantly contributes to pathogenesis. Matrix metalloproteinases (MMPs) are host endopeptidases capable of degrading a broad range of substrates, e.g. extracellular matrix proteins but also bioactive molecules. After activation by unsealing the catalytic site from the propeptide, the enzymes intervene in many physiological processes, e.g. morphogenesis, inflammation and wound repair. Their activity is fine-tuned by endogenous inhibitors, e.g. the tissue inhibitors of metalloproteinases (TIMPs). However, inappropriately regulated MMP activity is implicated in a variety of pathological conditions, such as uncontrolled inflammation, cancer, vascular and neurodegenerative disorders and autoimmune diseases. MMPs may also contribute to infection-related pathology, e.g. by favoring pathogen dissemination throughout the body and by opening barriers to immunoprivileged sites. In malaria, trypanosomiasis, leishmaniasis and toxoplasmosis, a common denominator is meningoencephalitis, characterized by the sequestration and/or migration of leukocytes and/or parasites across the blood-brain barrier (BBB), probably assisted by the proteolytic activity of MMPs. During its development inside the RBC, the parasite digests in its parasitophorous vacuole up to 80% of the host cell hemoglobin as a major nutrient source. During this catabolic process, toxic free heme is liberated and rapidly detoxified into a brownish insoluble crystal called hemozoin. Upon rupture of the schizont, hemozoin is released into the circulation and this malaria pigment catalyzes the production of reactive oxygen species and triggers immunomodulation linked to severe malaria pathology. In malaria, hemozoin is reported as an MMP-9 agonist. Based on the association of activated MMP-9 with several hemolytic diseases, e.g. experimental cerebral malaria, together with the resemblance of the hemopexin-domain of MMP-9 and the plasma hemopexin protein, the physiological scavenger of heme, it was intriguing to further decipher the link between hemozoin and MMP-9. Furthermore, hemozoin accumulation is observed in brain vessels of human malaria patients. By performing in vitro incubation experiments, it was shown that a direct interaction between ß-hematin or synthetic hemozoin, which is structurally identical to natural hemozoin, and the MMP-9 hemopexin domain provokes autocatalytic aminoterminal processing of the propeptide of MMP-9. The truncated enzyme was still catalytically inactive and protein sequencing by Edman degradation revealed that the ß-hematin-induced cleavage site was identical to the first cleavage site of MMP-3, a well-known activator of MMP-9 which cleaves the prodomain in two steps. This ß-hematin-mediated allosteric priming of proMMP-9 significantly enhanced the kinetics of its activation by the catalytic domain of MMP-3. The use of physiological concentrations of ß-hematin might imply some biological relevance for this process occurring in vivo, e.g. in cerebral malaria where disruption of the BBB is central to the pathogenesis. Although hemozoin was initially described as a metabolically inert waste product of the parasite, there is compelling evidence that this crystalloid pigment is involved in malaria-associated immunopathology. Hemozoin was observed to accumulate in bone marrow and to substantially contribute to severe malaria anemia that is caused by both loss and inadequate production of RBCs. In view of the hemozoin-induced priming of MMP-9 activation, the question was raised whether active MMP-9 might cleave and inactivate major erythropoietic growth factors, e.g. erythropoietin (Epo) and Growth Arrest Specific 6 (Gas6). No processing of these factors, however, was observed after overnight incubation with active MMP-9. On top of its function as a major MMP-9 endogenous inhibitor, TIMP-1 was documented as having erythroid-potentiating activity. By studying an experimental Plasmodium chabaudi (PcAS) infection in TIMP-1-/- mice, it became evident that deletion of TIMP-1 had no aggravating effect on the course of anemia. Taken together, these data suggest that both MMP-9 and TIMP-1 have no profound role in the regulation of erythropoietic processes. The molecular mechanisms through which hemozoin mediates its immunological effects are still not elucidated. This parasite-host interplay was suggested to involve Toll-like receptor 9 (TLR9), although it is still not clear whether hemozoin itself is sensed byTLR9, or if this crystalloid pigment functions as a vector that targets parasite DNA to TLR9. In addition, controversies exist on the significance of this pathogen recognition receptor in malaria immunological and pathogenic processes. To evaluate the in vivo role of TLR9 during malaria infection, a non-lethal PcAS infection was studied in genetically modified mice that lack TLR9. In comparison with commercial C57Bl/6 wild type (WT) control mice, these TLR9-/- mice initially showed an increased susceptibility phenotype, characterized by elevated acute and chronic parasitemia levels as well as a Th2-skewed antibody profile. However, these differences disappeared when WT and TLR9-/- mice derived from one intercross generation between both strains were compared. Although the TLR9-/- mice were originally received as being backcrossed onto C57Bl/6 for at least 10 generations, reassessment of the genetic background via single nucleotide polymorphism (SNP) analysis revealed that these TLR9-/- mice were definitely not congenic to C57Bl/6. Indeed, these knockout (KO) mice still carried large regions of 129 DNA, probably originating from the 129 stem cell donor in which the gene is mutated. The degree of C57Bl/6 DNA in the TLR9-/- mice was calculated to be 69%, indicating a maximum of two backcross generations to the C57Bl/6 inbred strain had occurred. Hence, the observed susceptibility phenotype against PcAS in the TLR9-/- mice was not induced by the null mutation, but was probably provoked by the heterogeneous genetic background since the 129 strain is reported to be susceptible for PcAS infection. The random shuffling of the 129 genes during meiosis might have caused the loss of phenotypic differences between the additionally backcrossed TLR9-/- mice and their matched WT controls. These findings illustrate the importance of studying gene function in congenic strains, i.e. strains that are, as a result of repeatedly backcrossing mice heterozygous for a mutation to an inbred stain, genetically identical to this inbred strain except for the targeted gene and its flanking region derived from the donor strain. This strategy will minimize phenotypic line differences due to background genes and thereby reduce the chance of misinterpretations. Furthermore, since most genes do not function in isolation but instead have to be considered in a multigenetic context, it is appropriate to analyze the effects of null mutations on different congenic genetic backgrounds. Solutions for the problem of the flanking genes are the conditional KO models as well as the use of co-isogenic lines (ES cells from donor strain with desired genetic background). Unfortunately, it has to be acknowledged that in many research papers on KO studies, only a sloppy description of the used mice is provided, which complicates rederivation of the identity of the mice and confounds reproducibility. This might explain the discrepancies, for example, on the role of TLR9 during a malaria infection. Hence, researchers need to be aware of the profound effects of the genetic background on gene function and we advocate that for all experiments designed with genetically modified mice, a detailed description on the origin and genetic background of both the control WT and the mutant strain is presented. In conclusion, MMPs may contribute to infection-related pathology due to their elevated and unbalanced proteolytic activity. For instance, we found that hemozoin is able to prime the activation of MMP-9, one of the main inflammatory MMPs. However, further elucidation of the regulation and functional roles played by MMPs/TIMPs during malaria may improve insights into immunopathogenic processes. Studies with MMP KO mice can help unravel gene function, yet it is important to evaluate KO phenotypes on a well-defined genetic background identical to the control mice. Indeed, the heterogeneity in the genetic background might be a confounding factor, for example, to explain the current controversies on the role of TLR9 in malaria infections. Hence, it is of critical importance to describe the used mice in full detail when publishing data of studies with genetically modified animals. <w:latentstyles deflockedstate="false" defunhidewhenused="true"  <w:lsdexception="" locked="false" priority="0" semihidden="false"  status: publishe

Zebrafish Skeleton Measurements using Image Analysis and Machine Learning Methods

The zebrafish is a model organism for biological studies on development and gene function. Our work aims at automating the detection of the cartilage skeleton and measuring several distances and angles to quantify its development following different experimental conditions