The anti-inflammatory peptide Catestatin blocks chemotaxis

Increased levels of the anti-inflammatory peptide Catestatin (CST), a cleavage product of the pro-hormone chromogranin A, correlate with less severe outcomes in hypertension, colitis, and diabetes. However, it is unknown how CST reduces the infiltration of monocytes and macrophages (Mϕs) in inflamed tissues. Here, it is reported that CST blocks leukocyte migration toward inflammatory chemokines. By in vitro and in vivo migration assays, it is shown that although CST itself is chemotactic, it blocks migration of monocytes and neutrophils to inflammatory attracting factor CC-chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 2 (CXCL2). Moreover, it directs CX3CR1+ Mϕs away from pancreatic islets. These findings suggest that the anti-inflammatory actions of CST are partly caused by its regulation of chemotaxis

Circulating Markers of Neutrophil Extracellular Traps Are of Prognostic Value in Patients With COVID-19

OBJECTIVE: The full spectrum of coronavirus disease 2019 (COVID-19) infection ranges from asymptomatic to acute respiratory distress syndrome, characterized by hyperinflammation and thrombotic microangiopathy. The pathogenic mechanisms are poorly understood, but emerging evidence suggest that excessive neutrophil extracellular trap (NET) formation plays a key role in COVID-19 disease progression. Here, we evaluate if circulating markers of NETs are associated with COVID-19 disease severity and clinical outcome, as well as to markers of inflammation and in vivo coagulation and fibrinolysis. Approach and Results: One hundred six patients with COVID-19 with moderate to severe disease were enrolled shortly after hospital admission and followed for 4 months. Acute and convalescent plasma samples as well as plasma samples from 30 healthy individuals were assessed for markers of NET formation: citrullinated histone H3, cell-free DNA, NE (neutrophil elastase). We found that plasma levels of NET markers were all elevated in patients with COVID-19 relative to healthy controls, were associated with respiratory support requirement and short-term mortality, and declined to those found in healthy individuals 4 months post-infection. Levels of the NET markers also correlated with white blood cells, neutrophils, inflammatory cytokines, and C-reactive protein, as well as to markers of in vivo coagulation, fibrinolysis, and endothelial damage. CONCLUSIONS: Our findings suggest a role of NETs in COVID-19 disease progression, implicating their contribution to an immunothrombotic state. Further, we observed an association between circulating markers of NET formation and clinical outcome, demonstrating a potential role of NET markers in clinical decision-making, as well as for NETs as targets for novel therapeutic interventions in COVID-19. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04402944, NCT04541979, NCT04445285, NCT04432987, NCT04402970

Ul Mira 14, Saint Petersburg 197101, Russia 6 St. Petersburg Hospital No. 31, Pr. Dinamo 3, Saint Petersburg

The spread of carbapenemase-producing Enterobacteriaceae is a global problem; however, no exact data on the epidemiology of carbapenemase in the Baltic countries and St. Petersburg area is available. We aimed to evaluate the epidemiology of carbapenemaseproducing Escherichia coli and Klebsiella pneumoniae in the Baltic States and St. Petersburg, Russia, and to compare the different methods for carbapenemase detection. From January to May 2012, all K. pneumoniae ( = 1983) and E. coli ( = 7774) clinical isolates from 20 institutions in Estonia, Latvia, Lithuania, and St. Petersburg, Russia were screened for carbapenem susceptibility. The IMP, VIM, GIM, NDM, KPC, and OXA-48 genes were detected using real-time PCR and the ability to hydrolyze ertapenem was determined using MALDI-TOF MS. Seventy-seven strains were found to be carbapenem nonsusceptible. From these, 15 K. pneumoniae strains hydrolyzed ertapenem and carried the NDM gene. All of these strains carried integron 1 and most carried integron 3 as well as genes of the CTX-M-1 group. No carbapenemase-producing E. coli or K. pneumoniae strains were found in Estonia, Latvia, or Lithuania; however, NDM-positive K. pneumoniae was present in the hospital in St. Petersburg, Russia. A MALDI-TOF MS-based assay is a suitable and cost-effective method for the initial confirmation of carbapenemase production

Macrophages and neutrophils in tissue homeostasis and recovery from ischemic injury

Neutrophils and macrophages have functions beyond protection against pathogens. The overall aim of the work presented in this thesis was to identify novel tasks for these innate immune cells in maintaining homeostasis.  In the studies presented here, we explored macrophage roles in tissue recovery from ischemic injury and post-natal tissue development, and the origin and recruitment mechanisms of pro-angiogenic neutrophils (PANs) to the site of ischemic injury. In Study I, it was shown that perivascular macrophages at sites of ischemic injury adopt characteristics of mural cell identity.  Combining genetic heritable fate mapping of macrophages with single-cell RNA-sequencing, we were able to demonstrate that macrophages downregulated the expression of myeloid markers, and upregulated those of mural cells. Depletion of macrophages during tissue healing demonstrated that macrophages also adopt important mural cell functions that are crucial for blood vessel maturation after ischemic injury.  In Study II, it was shown that perivascular macrophages form cuff-like structures around vessels in the ischemic muscle following ischemia. Using genetically modified mouse models, we showed that these macrophages regulate bloodflow in an inducible NO Synthase (iNOS)-dependent manner, and this could be therapeutically targeted to improve tissue healing through local delivery of plasmid-encoded C-X-C Motif Chemokine Ligand 12 (CXCL12).  In Study III, it was shown that recruitment of PANs to the site of ischemic injury is dependent on CD49d signalling, and the spleen contains a peripheral reservoir of PANs which is crucial for achieving adequate accumulation of PANs at the site of ischemic injury. We also showed that the release of splenic PANs in response to ischemic injury relies on sympathetic signalling and downregulation of CXCL12α in the splenic red pulp. In Study IV, it was shown that pancreatic tissue-resident macrophages are important for post-natal islet development, as depletion of macrophages using clodronate liposomes led to impaired glucose tolerance in adult mice. Further, neonatal infection with S. aureus led to reduced number of pancreatic macrophages and interfered with normal post-natal β cell development, leading to impaired glucose tolerance.   In summary, the work presented here expands our understanding on the various roles of macrophages during ischemic injury and tissue development, and significantly advances our understanding of the origins and recruitment mechanisms of pro-angiogenic neutrophils during ischemic injury.

Phagocytosis and Efferocytosis by Resident Macrophages in the Mouse Pancreas

The tissue microenvironment in the mouse pancreas has been shown to promote very different polarizations of resident macrophages with islet-resident macrophages displaying an inflammatory “M1” profile and macrophages in the exocrine tissue mostly displaying an alternatively activated “M2” profile. The impact of this polarization on tissue homeostasis and diabetes development is unclear. In this study, the ability of pancreas-resident macrophages to phagocyte bacterial and endogenous debris was investigated. Mouse endocrine and exocrine tissues were separated, and tissue-resident macrophages were isolated by magnetic immunolabeling. Isolated macrophages were subjected to flow cytometry for polarization markers and qPCR for phagocytosis-related genes. Functional in vitro investigations included phagocytosis and efferocytosis assays using pH-sensitive fluorescent bacterial particles and dead fluorescent neutrophils, respectively. Intravital confocal imaging of in situ phagocytosis and efferocytosis in the pancreas was used to confirm findings in vivo. Gene expression analysis revealed no significant overall difference in expression of most phagocytosis-related genes in islet-resident vs. exocrine-resident macrophages included in the analysis. In this study, pancreas-resident macrophages were shown to differ in their ability to phagocyte bacterial and endogenous debris depending on their microenvironment. This difference in abilities may be one of the factors polarizing islet-resident macrophages to an inflammatory state since phagocytosis has been found to imprint macrophage heterogeneity. It remains unclear if this difference has any implications in the development of islet dysfunction or autoimmunity

The anti-inflammatory peptide Catestatin blocks chemotaxis

Increased levels of the anti-inflammatory peptide Catestatin (CST), a cleavage product of the pro-hormone chromogranin A, correlate with less severe outcomes in hypertension, colitis, and diabetes. However, it is unknown how CST reduces the infiltration of monocytes and macrophages (M phi s) in inflamed tissues. Here, it is reported that CST blocks leukocyte migration toward inflammatory chemokines. By in vitro and in vivo migration assays, it is shown that although CST itself is chemotactic, it blocks migration of monocytes and neutrophils to inflammatory attracting factor CC-chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 2 (CXCL2). Moreover, it directs CX(3)CR1(+) M phi s away from pancreatic islets. These findings suggest that the anti-inflammatory actions of CST are partly caused by its regulation of chemotaxis

Macrophages upregulate mural cell-like markers and support healing of ischemic injury by adopting functions important for vascular support

Sterile inflammation after injury is important for tissue restoration. In injured human and mouse tissues, macrophages were recently found to accumulate perivascularly. This study investigates if macrophages adopt a mural cell phenotype important for restoration after ischemic injury. Single-cell RNA sequencing of fate-mapped macrophages from ischemic mouse muscles demonstrates a macrophage-toward-mural cell switch of a subpopulation of macrophages with downregulated myeloid cell genes and upregulated mural cell genes, including PDGFRβ. This observation was further strengthened when including unspliced transcripts in the analysis. The macrophage switch was proven functionally relevant, as induction of macrophage-specific PDGFRβ deficiency prevented their perivascular macrophage phenotype, impaired vessel maturation and increased vessel leakiness, which ultimately reduced limb function. In conclusion, macrophages in adult ischemic tissue were demonstrated to undergo a cellular program to morphologically, transcriptomically and functionally resemble mural cells while weakening their macrophage identity. The macrophage-to-mural cell-like phenotypic switch is crucial for restoring tissue function and warrants further exploration as a potential target for immunotherapies to enhance healing.De två första författarna delar förstaförfattarskapet</p

Oral administration of CXCL12-expressing Limosilactobacillus reuteri improves colitis by local immunomodulatory actions in preclinical models

Treatments of colitis, inflammation of the intestine, rely on induction of immune suppression associated with systemic adverse events, including recurrent infections. This treatment strategy is specifically problematic in the increasing population of patients with cancer with immune checkpoint inhibitor (ICI)-induced colitis, as immune suppression also interferes with the ICI-treatment response. Thus, there is a need for local-acting treatments that reduce inflammation and enhance intestinal healing. Here, we investigated the effect and safety of bacterial delivery of short-lived immunomodulating chemokines to the inflamed intestine in mice with colitis. Colitis was induced by dextran sulfate sodium (DSS) alone or in combination with ICI (anti-PD1 and anti-CTLA-4), and Limosilactobacillus reuteri R2LC (L. reuteri R2LC) genetically modified to express the chemokine CXCL12-1 alpha (R2LC_CXCL12, emilimogene sigulactibac) was given perorally. In addition, the pharmacology and safety of the formulated drug candidate, ILP100-Oral, were evaluated in rabbits. Peroral CXCL12-producing L. reuteri R2LC significantly improved colitis symptoms already after 2 days in mice with overt DSS and ICI-induced colitis, which in benchmarking experiments was demonstrated to be superior to treatments with anti-TNF-alpha, anti-alpha 4 beta 7, and corticosteroids. The mechanism of action involved chemokine delivery to Peyer's patches (PPs), confirmed by local CXCR4 signaling, and increased numbers of colonic, regulatory immune cells expressing IL-10 and TGF-beta 1. No systemic exposure or engraftment could be detected in mice, and product feasibility, pharmacology, and safety were confirmed in rabbits. In conclusion, peroral CXCL12-producing L. reuteri R2LC efficiently ameliorates colitis, enhances mucosal healing, and has a favorable safety profile. NEW &amp; NOTEWORTHY Colitis symptoms are efficiently reduced by peroral administration of probiotic bacteria genetically modified to deliver CXCL12 locally to the inflamed intestine in several mouse models.Authors in the list of papers of Emelie Öhnstedt's thesis: Emelie Öhnstedt, Cristian Doñas, Kristel Parv, Yanhong Pang, Hava Lofton Tomenius, Macarena Carrasco López, Venkata Ram Gannavarapu, Peter Frank, Margareth Jorvid, Stefan Roos, Evelina Vågesjö, Mia Phillipson</p

Differences in Extended-Spectrum Beta-Lactamase Producing Escherichia coli Virulence Factor Genes in the Baltic Sea Region

The aim of this study was to compare the prevalence of different virulence factor (VF) genes in extended-spectrum beta-lactamase (ESBL) producing Escherichia coli strains isolated from the Baltic Sea region. A total of 432 strains of phenotypically ESBL positive E. coli were collected from 20 institutions located in Estonia, Latvia, Lithuania, and the region of St. Petersburg in Russia from January to May 2012 and analyzed for phylogenetic group and prevalence of 23 VF genes. The strains were collected from clinical material (urine, blood, wound, and respiratory tract). Bacterial isolates were compared according to phylogenetic group, clinical material, and geographical origin. Most of the VF genes were concentrated within phylogenetic group B2 and/or D. When comparing strains isolated from different countries, it was found that strains originating from Estonia and Latvia belonged mainly to group B2 and strains from Lithuania and Russia mainly to groups B2 and D. The P-fimbrial adhesin gene papEF was more prevalent in Russian strains, colicin gene cvaC in Lithuanian strains, and capsular gene kpsMTII in Latvian strains; serum resistant gene traT was less prevalent in Estonian strains. The regional differences of VF genes remained statistically significant after taking into account the phylogenetic distribution in the countries