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 CXCL12-1α (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-α, anti-α4ß7, 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-ß1. 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 & 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.

Perivascular Macrophages Regulate Blood Flow Following Tissue Damage.

[Figure: see text].

Accelerated wound healing in mice by on-site production and delivery of CXCL12 by transformed lactic acid bacteria.

Impaired wound closure is a growing medical problem associated with metabolic diseases and aging. Immune cells play important roles in wound healing by following instructions from the microenvironment. Here, we developed a technology to bioengineer the wound microenvironment and enhance healing abilities of the immune cells. This resulted in strongly accelerated wound healing and was achieved by transforming Lactobacilli with a plasmid encoding CXCL12. CXCL12-delivering bacteria administrated topically to wounds in mice efficiently enhanced wound closure by increasing proliferation of dermal cells and macrophages, and led to increased TGF-ß expression in macrophages. Bacteria-produced lactic acid reduced the local pH, which inhibited the peptidase CD26 and consequently enhanced the availability of bioactive CXCL12. Importantly, treatment with CXCL12-delivering Lactobacilli also improved wound closure in mice with hyperglycemia or peripheral ischemia, conditions associated with chronic wounds, and in a human skin wound model. Further, initial safety studies demonstrated that the topically applied transformed bacteria exerted effects restricted to the wound, as neither bacteria nor the chemokine produced could be detected in systemic circulation. Development of drugs accelerating wound healing is limited by the proteolytic nature of wounds. Our technology overcomes this by on-site chemokine production and reduced degradation, which together ensure prolonged chemokine bioavailability that instructed local immune cells and enhanced wound healing.

Identification and characterization of VEGF-A-responsive neutrophils expressing CD49d, VEGFR1, and CXCR4 in mice and humans.

Vascular endothelial growth factor A (VEGF-A) is upregulated during hypoxia and is the major regulator of angiogenesis. VEGF-A expression has also been found to recruit myeloid cells to ischemic tissues where they contribute to angiogenesis. This study investigates the mechanisms underlying neutrophil recruitment to VEGF-A as well as the characteristics of these neutrophils. A previously undefined circulating subset of neutrophils shown to be CD49d(+)VEGFR1(high)CXCR4(high) was identified in mice and humans. By using chimeric mice with impaired VEGF receptor 1 (VEGFR1) or VEGFR2 signaling (Flt-1tk(-/-), tsad(-/-)), we found that parallel activation of VEGFR1 on neutrophils and VEGFR2 on endothelial cells was required for VEGF-A-induced recruitment of circulating neutrophils to tissue. Intravital microscopy of mouse microcirculation revealed that neutrophil recruitment by VEGF-A versus by the chemokine macrophage inflammatory protein 2 (MIP-2 [CXCL2]) involved the same steps of the recruitment cascade but that an additional neutrophil integrin (eg, VLA-4 [CD49d/CD29]) played a crucial role in neutrophil crawling and emigration to VEGF-A. Isolated CD49d(+) neutrophils featured increased chemokinesis but not chemotaxis compared with CD49d(-) neutrophils in the presence of VEGF-A. Finally, by targeting the integrin α4 subunit (CD49d) in a transplantation-based angiogenesis model that used avascular pancreatic islets transplanted to striated muscle, we demonstrated that inhibiting the recruitment of circulating proangiogenic neutrophils to hypoxic tissue impairs vessel neoformation. Thus, angiogenesis can be modulated by targeting cell-surface receptors specifically involved in VEGF-A-dependent recruitment of proangiogenic neutrophils without compromising recruitment of the neutrophil population involved in the immune response to pathogens.

VEGF-A recruits a proangiogenic MMP-9-delivering neutrophil subset that induces angiogenesis in transplanted hypoxic tissue.

Recruitment and retention of leukocytes at a site of blood vessel growth are crucial for proper angiogenesis and subsequent tissue perfusion. Although critical for many aspects of regenerative medicine, the mechanisms of leukocyte recruitment to and actions at sites of angiogenesis are not fully understood. In this study, we investigated the signals attracting leukocytes to avascular transplanted pancreatic islets and leukocyte actions at the engraftment site. Expression of the angiogenic stimulus VEGF-A by mouse pancreatic islets was elevated shortly after syngeneic transplantation to muscle. High levels of leukocytes, predominantly CD11b(+)/Gr-1(+)/CXCR4(hi) neutrophils, were observed at the site of engraftment, whereas VEGF-A-deficient islets recruited only half of the amount of leukocytes when transplanted. Acute VEGF-A exposure of muscle increased leukocyte extravasation but not the levels of SDF-1α. VEGF-A-recruited neutrophils expressed 10 times higher amounts of MMP-9 than neutrophils recruited to an inflammatory stimulus. Revascularization of islets transplanted to MMP-9-deficient mice was impaired because blood vessels initially failed to penetrate grafts, and after 2 weeks vascularity was still disturbed. This study demonstrates that VEGF-A recruits a proangiogenic circulating subset of CD11b(+)/Gr-1(+) neutrophils that are CXCR4(hi) and deliver large amounts of the effector protein MMP-9, required for islet revascularization and functional integration after transplantation.

Acute sleep deprivation in healthy young men: impact on population diversity and function of circulating neutrophils.

Lack of sleep greatly affects our immune system. The present study investigates the acute effects of total sleep deprivation on blood neutrophils, the most abundant immune cell in our circulation and the first cell type recruited to sites of infection. Thus, the population diversity and function of circulating neutrophils were compared in healthy young men following one night of total sleep deprivation (TSD) or after 8h regular sleep. We found that neutrophil counts were elevated after nocturnal wakefulness (2.0 ± 0.2 × 10(9)/l vs. 2.6 ± 0.2 × 10(9)/l, sleep vs. TSD, respectively) and the population contained more immature CD16(dim)/CD62L(bright) cells (0.11 ± 0.040 × 10(9)/l [5.5 ± 1.1%] vs. 0.26 ± 0.020 × 10(9)/l [9.9 ± 1.4%]). As the rise in numbers of circulating mature CD16(bright)/CD62L(bright) neutrophils was less pronounced, the fraction of this subpopulation showed a significant decrease (1.8 ± 0.15 × 10(9)/l [88 ± 1.8%] vs. 2.1 ± 0.12 × 10(9)/l [82 ± 2.8%]). The surface expression of receptors regulating mobilization of neutrophils from bone marrow was decreased (CXCR4 and CD49d on immature neutrophils; CXCR2 on mature neutrophils). The receptor CXCR2 is also involved in the production of reactive oxygen species (ROS), and in line with this, total neutrophils produced less ROS. In addition, following sleep loss, circulating neutrophils exhibited enhanced surface levels of CD11b, which indicates enhanced granular fusion and concomitant protein translocation to the membrane. Our findings demonstrate that sleep loss exerts significant effects on population diversity and function of circulating neutrophils in healthy men. To which extent these changes could explain as to why people with poor sleep patterns are more susceptible to infections warrants further investigation.

Corpora cavernosa fibroblasts mediate penile erection.

Penile erection is mediated by the corpora cavernosa, a trabecular-like vascular bed that enlarges upon vasodilation, but its regulation is not completely understood. Here, we show that perivascular fibroblasts in the corpora cavernosa support vasodilation by reducing norepinephrine availability. The effect on penile blood flow depends on the number of fibroblasts, which is regulated by erectile activity. Erection dynamically alters the positional arrangement of fibroblasts, temporarily down-regulating Notch signaling. Inhibition of Notch increases fibroblast numbers and consequently raises penile blood flow. Continuous Notch activation lowers fibroblast numbers and reduces penile blood perfusion. Recurrent erections stimulate fibroblast proliferation and limit vasoconstriction, whereas aging reduces the number of fibroblasts and lowers penile blood flow. Our findings reveal adaptive, erectile activity-dependent modulation of penile blood flow by fibroblasts.

Engineered bacteria to accelerate wound healing: an adaptive, randomised, double-blind, placebo-controlled, first-in-human phase 1 trial.

Background: Impaired wound healing is a growing medical problem and very few approved drugs with documented clinical efficacy are available. CXCL12-expressing lactic acid bacteria, Limosilactobacillus reuteri (ILP100-Topical), has been demonstrated to accelerate wound healing in controlled preclinical models. In this first-in-human study, the primary objective was to determine safety and tolerability of the drug candidate ILP100-Topical, while secondary objectives included assessments of clinical and biologic effects on wound healing by traditionally accepted methods and explorative and traceable assessments. Methods: SITU-SAFE is an adaptive, randomised, double-blind, placebo-controlled, first-in-human phase 1 trial (EudraCT 2019-000680-24) consisting of a single (SAD) and a multiple ascending dose (MAD) part of three dose cohorts each. The study was performed at the Phase 1 Unit, Uppsala University Hospital, Uppsala, Sweden. Data in this article were collected between Sep 20th, 2019 and Oct 20th 2021. In total 240 wounds were induced on the upper arms in 36 healthy volunteers. SAD: 12 participants, 4 wounds (2/arm), MAD: 24 participants, 8 wounds (4/arm). Wounds in each participant were randomised to treatment with placebo/saline or ILP100-Topical. Findings: In all individuals and doses, ILP100-Topical was safe and well-tolerated with no systemic exposure. A combined cohort analysis showed a significantly larger proportion of healed wounds (p = 0.020) on Day 32 by multi-dosing of ILP100-Topical when compared to saline/placebo (76% (73/96) and 59% (57/96) healed wounds, respectively). In addition, time to first registered healing was shortened by 6 days on average, and by 10 days at highest dose. ILP100-Topical increased the density of CXCL12+ cells in the wounds and local wound blood perfusion. Interpretation: The favourable safety profile and observed effects on wound healing support continued clinical development of ILP100-Topical for the treatment of complicated wounds in patients. Funding: Ilya Pharma AB (Sponsor), H2020 SME Instrument Phase II (#804438), Knut and Alice Wallenberg foundation.

Vascular adaptation to a dysfunctional endothelium as a consequence of Shb deficiency.

Vascular endothelial growth factor (VEGF)-A regulates angiogenesis, vascular morphology and permeability by signaling through its receptor VEGFR-2. The Shb adapter protein has previously been found to relay certain VEGFR-2 dependent signals and consequently vascular physiology and structure was assessed in Shb knockout mice. X-ray computed tomography of vessels larger than 24 µm diameter (micro-CT) after contrast injection revealed an increased frequency of 48-96 µm arterioles in the hindlimb calf muscle in Shb knockout mice. Intravital microscopy of the cremaster muscle demonstrated a less regular vasculature with fewer branch points and increased vessel tortuosity, changes that led to an increased blood flow velocity. Reduced in vivo angiogenesis was observed in Shb knockout Matrigel™ plugs. Unlike the wild-type situation, VEGF-A did not provoke a dissociation of VE-cadherin from adherens junctions in Shb knockout venules. The reduced angiogenesis and altered properties of junctions had consequences for two patho-physiological responses to arterial occlusion: vascular permeability was reduced in the Shb knockout cremaster muscle after ligation of one supplying artery and heat-induced blood flow determined by Laser-Doppler measurements was decreased in the hindlimb after ligation of the femoral artery. Consequently, the Shb knockout mouse exhibited structural and functional (angiogenesis and vascular permeability) vascular abnormalities that have implications for understanding the function of VEGF-A under physiological conditions.

Accelerated Wound Healing in Minipigs by On-Site Production and Delivery of CXCL12 by Transformed Lactic Acid Bacteria.

Non-healing wounds are a growing medical problem and result in considerable suffering. The lack of pharmaceutical treatment options reflects the multistep wound healing process, and the complexity of both translation and assessment of treatment efficacy. We previously demonstrated accelerated healing of full-thickness wounds in mice following topical application of the probiotic bacteria Limosilactobacillus reuteri R2LC transformed to express CXCL12. In this study, safety and biological effects of a freeze-dried formulation of CXCL12-producing L. reuteri (ILP100) were investigated in induced full-thickness wounds in minipigs, and different wound healing evaluation methods (macroscopic, planimetry, 2D-photographs, 3D-scanning, ultrasound) were compared. We found that treatment with ILP100 was safe and accelerated healing, as granulation tissue filled wound cavities 1 day faster in treated compared to untreated/placebo-treated wounds. Furthermore, evaluation using planimetry resulted in 1.5 days faster healing than using 2D photographs of the same wounds, whereas the areas measured using 2D photographs were smaller compared to those obtained from 3D scans accounting for surface curvatures, whereas ultrasound imaging enabled detailed detection of thin epithelial layers. In conclusion, topical administration of the drug candidate ILP100 warrants further clinical development as it was proven to be safe and to accelerate healing using different evaluation methods in minipigs.

In Vivo and In Vitro Detection of Luminescent and Fluorescent Lactobacillus reuteri and Application of Red Fluorescent mCherry for Assessing Plasmid Persistence.

Lactobacillus reuteri is a symbiont that inhabits the gastrointestinal (GI) tract of mammals, and several strains are used as probiotics. After introduction of probiotic strains in a complex ecosystem like the GI tract, keeping track of them is a challenge. The main objectives of this study were to introduce reporter proteins that would enable in vivo and in vitro detection of L. reuteri and increase knowledge about its interactions with the host. We describe for the first time cloning of codon-optimized reporter genes encoding click beetle red luciferase (CBRluc) and red fluorescent protein mCherry in L. reuteri strains ATCC PTA 6475 and R2LC. The plasmid persistence of mCherry-expressing lactobacilli was evaluated by both flow cytometry (FCM) and conventional plate count (PC), and the plasmid loss rates measured by FCM were lower overall than those determined by PC. Neutralization of pH and longer induction duration significantly improved the mCherry signal. The persistency, dose-dependent signal intensity and localization of the recombinant bacteria in the GI tract of mice were studied with an in vivo imaging system (IVIS), which allowed us to detect fluorescence from 6475-CBRluc-mCherry given at a dose of 1×1010 CFU and luminescence signals at doses ranging from 1×105 to 1×1010 CFU. Both 6475-CBRluc-mCherry and R2LC-CBRluc were localized in the colon 1 and 2 h after ingestion, but the majority of the latter were still found in the stomach, possibly reflecting niche specificity for R2LC. Finally, an in vitro experiment showed that mCherry-producing R2LC adhered efficiently to the intra cellular junctions of cultured IPEC-J2 cells. In conclusion, the two reporter genes CBRluc and mCherry were shown to be suitable markers for biophotonic imaging (BPI) of L. reuteri and may provide useful tools for future studies of in vivo and in vitro interactions between the bacteria and the host.

Immunological shielding by induced recruitment of regulatory T-lymphocytes delays rejection of islets transplanted in muscle.

The only clinically available curative treatment of type 1 diabetes mellitus is replacement of the pancreatic islets by allogeneic transplantation, which requires immunosuppressive therapies. Regimens used today are associated with serious adverse effects and impaired islet engraftment and function. The aim of the current study was to induce local immune privilege by accumulating immune-suppressive regulatory T-lymphocytes (Tregs) at the site of intramuscular islet transplantation to reduce the need of immunosuppressive therapy during engraftment. Islets were cotransplanted with a plasmid encoding the chemokine CCL22 into the muscle of MHC-mismatched mice, after which pCCL22 expression and leukocyte recruitment were studied in parallel with graft functionality. Myocyte pCCL22 expression and secretion resulted in local accumulation of Tregs. When islets were cotransplanted with pCCL22, significantly fewer effector T-lymphocytes were observed in close proximity to the islets, leading to delayed graft rejection. As a result, diabetic recipients cotransplanted with islets and pCCL22 intramuscularly became normoglycemic for 10 consecutive days, while grafts cotransplanted with control plasmid were rejected immediately, leaving recipients severely hyperglycemic. Here we propose a simple method to initially shield MHC-mismatched islets by the recruitment of endogenous Tregs during engraftment in order to improve early islet survival. Using this approach, the very high doses of systemic immunosuppression used initially following transplantation can thereby be avoided.

Acute sleep deprivation increases serum levels of neuron-specific enolase (NSE) and S100 calcium binding protein B (S-100B) in healthy young men.

STUDY OBJECTIVES: To investigate whether total sleep deprivation (TSD) affects circulating concentrations of neuron-specific enolase (NSE) and S100 calcium binding protein B (S-100B) in humans. These factors are usually found in the cytoplasm of neurons and glia cells. Increasing concentrations of these factors in blood may be therefore indicative for either neuronal damage, impaired blood brain barrier function, or both. In addition, amyloid ß (Aß) peptides 1-42 and 1-40 were measured in plasma to calculate their ratio. A reduced plasma ratio of Aß peptides 1-42 to 1-40 is considered an indirect measure of increased deposition of Aß 1-42 peptide in the brain. DESIGN: Subjects participated in two conditions (including either 8-h of nocturnal sleep [22:30-06:30] or TSD). Fasting blood samples were drawn before and after sleep interventions (19:30 and 07:30, respectively). SETTING: Sleep laboratory. PARTICIPANTS: 15 healthy young men. RESULTS: TSD increased morning serum levels of NSE (P = 0.002) and S-100B (P = 0.02) by approximately 20%, compared with values obtained after a night of sleep. In contrast, the ratio of Aß peptides 1-42 to 1-40 did not differ between the sleep interventions. CONCLUSIONS: Future studies in which both serum and cerebrospinal fluid are sampled after sleep loss should elucidate whether the increase in serum neuron-specific enolase and S100 calcium binding protein B is primarily caused by neuronal damage, impaired blood brain barrier function, or is just a consequence of increased gene expression in non-neuronal cells, such as leukocytes.

Aberrant association between vascular endothelial growth factor receptor-2 and VE-cadherin in response to vascular endothelial growth factor-a in Shb-deficient lung endothelial cells.

Vascular permeability is a hallmark response to the main angiogenic factor VEGF-A and we have previously described a reduction of this response in Shb knockout mice. To characterize the molecular mechanisms responsible for this effect, endothelial cells were isolated from lungs and analyzed in vitro. Shb deficient endothelial cells exhibited less migration in a scratch wound-healing assay both under basal conditions and after vascular endothelial growth factor-A (VEGF-A) stimulation, suggesting a functional impairment of these cells in vitro. Staining for VE-cadherin and vascular endothelial growth factor receptor-2 (VEGFR-2) showed co-localization in adherens junctions and in intracellular sites such as the perinuclear region in wild-type and Shb knockout cells. VEGF-A decreased the VE-cadherin/VEGFR-2 co-localization in membrane structures resembling adherens junctions in wild-type cells whereas no such response was noted in the Shb knockout cells. VE-cadherin/VEGFR-2 co-localization was also recorded using spinning-disk confocal microscopy and VEGF-A caused a reduced association in the wild-type cells whereas the opposite pattern was observed in the Shb knockout cells. The latter expressed slightly more of cell surface VEGFR-2. VEGF-A stimulated extracellular-signal regulated kinase, Akt and Rac1 activities in the wild-type cells whereas no such responses were noted in the knockout cells. We conclude that aberrant signaling characteristics with respect to ERK, Akt and Rac1 are likely explanations for the observed altered pattern of VE-cadherin/VEGFR-2 association. The latter is important for understanding the reduced in vivo vascular permeability response in Shb knockout mice, a phenomenon that has patho-physiological relevance.