Signal-regulatory protein α (SIRPα) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRPα and the "don't eat me" ligand CD47 and as a ...marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRPα (hSIRPα)-specific nanobodies (Nbs). We identified high-affinity Nbs targeting the hSIRPα/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis. For non-invasive
imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography in novel hSIRPα/hCD47 knock-in mice, we demonstrated the applicability of
Cu-hSIRPα-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRPα-Nbs presented in this study have potential as versatile theranostic probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for
stratification and monitoring of individual responses during cancer immunotherapies.
Organ dysfunction is a major concern in sepsis pathophysiology and contributes to its high mortality rate. Neutrophil extracellular traps (NETs) have been implicated in endothelial damage and take ...part in the pathogenesis of organ dysfunction in several conditions. NETs also have an important role in counteracting invading microorganisms during infection. The aim of this study was to evaluate systemic NETs formation, their participation in host bacterial clearance and their contribution to organ dysfunction in sepsis. C57Bl/6 mice were subjected to endotoxic shock or a polymicrobial sepsis model induced by cecal ligation and puncture (CLP). The involvement of cf-DNA/NETs in the physiopathology of sepsis was evaluated through NETs degradation by rhDNase. This treatment was also associated with a broad-spectrum antibiotic treatment (ertapenem) in mice after CLP. CLP or endotoxin administration induced a significant increase in the serum concentrations of NETs. The increase in CLP-induced NETs was sustained over a period of 3 to 24 h after surgery in mice and was not inhibited by the antibiotic treatment. Systemic rhDNase treatment reduced serum NETs and increased the bacterial load in non-antibiotic-treated septic mice. rhDNase plus antibiotics attenuated sepsis-induced organ damage and improved the survival rate. The correlation between the presence of NETs in peripheral blood and organ dysfunction was evaluated in 31 septic patients. Higher cf-DNA concentrations were detected in septic patients in comparison with healthy controls, and levels were correlated with sepsis severity and organ dysfunction. In conclusion, cf-DNA/NETs are formed during sepsis and are associated with sepsis severity. In the experimental setting, the degradation of NETs by rhDNase attenuates organ damage only when combined with antibiotics, confirming that NETs take part in sepsis pathogenesis. Altogether, our results suggest that NETs are important for host bacterial control and are relevant actors in the pathogenesis of sepsis.
BackgroundAlthough Immune checkpoint inhibitors (ICI)-targeting therapies have revolutionized the treatment of cancer, several tumors do not respond to those therapies. Preclinical and clinical ...evidences suggest that STING is a promising target to improve the immunogenicity of tumors, turning them responsive to ICI, and enhancing anti-tumor response. DMXAA failed to show efficacy in clinical trials, despite its encouraging anti-tumor response in preclinical phase, highlighting the need of accurate translational preclinical models. On top of the specificity barrier reported for STING-targeting agents, the heterogeneity of STING variants and their variability in the response to STING-targeting therapies, brings another level of complexity in preclinical evaluation of anti-STING therapies. Here, we report the generation of STING humanized (hSTING) mouse models enabling the in vivo assessment of STING-targeting agents.MethodsHuman STING variants show a high heterogenicity and population stratification. Different variants, and isoforms, respond differently to STING agonists. We developed mouse models expressing the main human STING variants and isoforms found in the population to recapitulate this complexity. Human STING was inserted by knock-in at the endogenous locus to enable a physiological expression pattern of STING while invalidating the mouse gene. Herein, we will focus on the human STING full length H323 model.ResultsT and B lymphocytes, NK, DCs and monocytes frequence in the spleen, bone marrow and blood were found to be similar in hSTING and WT mice, suggesting that the humanization of STING did not alter the immune cell distribution in these compartments. These cells express human STING, while no expression of mouse STING was observed. Splenocytes isolated from hSTING and WT mice produced IL-6 and IFN-γ upon activation with 2'3'-cGAMP, a cyclic dinucleotide with activity towards both mouse and human STING. Similarly, a mouse and human STING agonist induced the activation of DCs in both hSTING and WT mice, as observed by the increased expression of CD80/CD86 on DCs ex vivo and in vivo. Moreover, systemic production of IL-6, IFN-γ and TNF-α in response to this STING agonist was observed and suggest that human STING is functional in hSTING mice. As expected, hSTING mice did not respond to activation with DMXAA in vivo, whereas this agonist induced the systemic production of cytokines and activation of DCs in WT mice.ConclusionsThe novel hSTING model described here supports the assessment of human STING-targeting agents in immuno-oncology and inflammation. Intercrosses of this model with ICI humanized models could support the assessment of combination therapies
Pathogen recognition and triggering of the inflammatory response following infection in mammals depend mainly on Toll-like and Nod-like receptors. Here, we evaluated the role of Nod1, Nod2 and ...MyD88-dependent signaling in the chemokine production and neutrophil recruitment to the infectious site during sepsis induced by cecal ligation and puncture (CLP) in C57Bl/6 mice. We demonstrate that Nod1 and Nod2 are not involved in the release of chemokines and recruitment of neutrophils to the infectious site during CLP-induced septic peritonitis because these events were similar in wild-type, Nod1-, Nod2-, Nod1/Nod2- and Rip2-deficient mice. Consequently, the local and systemic bacterial loads were not altered. Accordingly, neither Nod1 nor Nod2 was involved in the production of the circulating cytokines and in the accumulation of leukocytes in the lungs. By contrast, we showed that MyD88-dependent signaling is crucial for the establishment of the local inflammatory response during CLP-induced sepsis. MyD88-deficient mice were susceptible to sepsis because of an impaired local production of chemokines and defective neutrophil recruitment to the infection site. Altogether, these data show that Nod1, Nod2 and Rip2 are not required for local chemokine production and neutrophil recruitment during CLP-induced sepsis, and they reinforce the importance of MyD88-dependent signaling for initiation of a protective host response.
Sepsis, an overwhelming inflammatory response syndrome secondary to infection, is one of the costliest and deadliest medical conditions worldwide. Neutrophils are classically considered to be ...essential players in the host defense against invading pathogens. However, several investigations have shown that impairment of neutrophil migration to the site of infection, also referred to as neutrophil paralysis, occurs during severe sepsis, resulting in an inability of the host to contain and eliminate the infection. On the other hand, the neutrophil antibacterial arsenal contributes to tissue damage and the development of organ dysfunction during sepsis. In this review, we provide an overview of the main events in which neutrophils play a beneficial or deleterious role in the outcome of sepsis.
BackgroundT-cell engagers have proved to be a promising therapeutic strategy in immunotherapy, for redirecting T cells activity against tumor cells. To facilitate the preclinical assessment of novel ...T-cell engagers and their translatability, we have developed an immunocompetent CD3 epsilon N-terminal epitope humanized mouse model.MethodsThis model was developed to express the human epitope of the CD3 epsilon chain, which is recognized by approximately 70% of the T-cell engagers (clone SP34). The rest of the extracellular domain was kept from mouse origin to preserve the amino acids involved in the interaction with CD3 gamma and delta. Similarly, the transmembrane domains and the intracellular domains where kept murine to enable salt bridges interaction, interaction with the CD3 zeta and the signaling into mouse cells.ResultsT cells from CD3 epsilon epitope humanized mice are found in comparable frequency in spleen, blood and bone marrow from WT mice. B cells, monocytes, dendritic cells and NK frequencies are also similar to the frequencies of these cell types in WT mice, suggesting that the humanization of the epitope of CD3 epsilon did not alter the immune cells distribution in these mice. Activation of T cells with antibodies targeting human CD3 (clone SP34) induced CD4 and CD8 T cell proliferation, as well as production of IL-2 and IFN-gamma. The CD3 functionality was demonstrated in vitro by the ability of B cells to produce IgM upon activation of T cells, suggesting a proper cooperation between T and B cells. Additionally, a first class of T-cell engagers targeting both human CD3 and a tumoral antigen, induced tumor cell lysis of MC38-Ag in a concentration-dependent manner. A second class of T cell engagers, also targeting CD3 and a tumoral antigen, showed an anti-tumor effect in vivo, and this effect was also shown to be dose-dependent.ConclusionsThese data suggest that the CD3 epsilon N-terminal epitope humanized mouse model enables the assessment of efficacy and mechanism of action of T-cell engagers.This model is currently being intercrossed with immunostimulatory humanized mouse models to provide new opportunities for assessment of bi-specific antibodies targeting the CD3 and immunostimulatory molecules. This model is the first generation of a broader program aiming at developping a Pan CD3 humanized model, where the gamma, delta and epsilon chains of the CD3 complex will be humanized. The Pan CD3 humanized mice are currently being investigated for immune responses and would provide a broader tool for assessment of T-cell engagers.
In autoimmune diseases, autoreactive B cells comprise only the 0.1-0.5% of total circulating B cells. However, current first-line treatments rely on non-specific and general suppression of the immune ...system, exposing patients to severe side effects. For this reason, identification of targeted therapies for autoimmune diseases is an unmet clinical need.
Here, we designed a novel class of immunotherapeutic molecules, Bi-specific AutoAntigen-T cell Engagers (BiAATEs), as a potential approach for targeting the small subset of autoreactive B cells. To test this approach, we focused on a prototype autoimmune disease of the kidney, membranous nephropathy (MN), in which phospholipase A
receptor (PLA
R) serves as primary nephritogenic antigen. Specifically, we developed a BiAATE consisting of the immunodominant Cysteine-Rich (CysR) domain of PLA
R and the single-chain variable fragment (scFv) of an antibody against the T cell antigen CD3, connected by a small flexible linker.
BiAATE creates an immunological synapse between autoreactive B cells bearing an CysR-specific surface Ig
and T cells.
, the BiAATE successfully induced T cell-dependent depletion of PLA
R-specific B cells isolated form MN patients, sparing normal B cells. Systemic administration of BiAATE to mice transgenic for human CD3 reduced anti-PLA
R antibody levels following active immunization with PLA
R.
Should this approach be confirmed for other autoimmune diseases, BiAATEs could represent a promising off-the-shelf therapy for precision medicine in virtually all antibody-mediated autoimmune diseases for which the pathogenic autoantigen is known, leading to a paradigm shift in the treatment of these diseases.
Sepsis is a systemic inflammatory response resulting from the inability of the host to contain the infection locally. Previously, we demonstrated that during severe sepsis there is a marked failure ...of neutrophil migration to the infection site, which contributes to dissemination of infection, resulting in high mortality. IL-17 plays an important role in neutrophil recruitment. Herein, we investigated the role of IL-17R signaling in polymicrobial sepsis induced by cecal ligation and puncture (CLP). It was observed that IL-17R-deficient mice, subjected to CLP-induced non-severe sepsis, show reduced neutrophil recruitment into the peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared with C57BL/6 littermates. As a consequence, the mice showed an increased mortality rate. The ability of IL-17 to induce neutrophil migration was demonstrated in vivo and in vitro. Beside its role in neutrophil recruitment to the infection focus, IL-17 enhanced the microbicidal activity of the migrating neutrophils by a mechanism dependent on NO. Therefore, IL-17 plays a critical role in host protection during polymicrobial sepsis.
Sepsis is a systemic inflammatory condition following bacterial infection with a high mortality rate and limited therapeutic options. Here we show that interleukin-33 (IL-33) reduces mortality in ...mice with experimental sepsis from cecal ligation and puncture (CLP). IL-33-treated mice developed increased neutrophil influx into the peritoneal cavity and more efficient bacterial clearance than untreated mice. IL-33 reduced the systemic but not the local proinflammatory response, and it did not induce a T helper type 1 (TH1) to TH2 shift. The chemokine receptor CXCR2 is crucial for recruitment of neutrophils from the circulation to the site of infection. Activation of Toll-like receptors (TLRs) in neutrophils downregulates CXCR2 expression and impairs neutrophil migration. We show here that IL-33 prevents the downregulation of CXCR2 and inhibition of chemotaxis induced by the activation of TLR4 in mouse and human neutrophils. Furthermore, we show that IL-33 reverses the TLR4-induced reduction of CXCR2 expression in neutrophils via the inhibition of expression of G protein-coupled receptor kinase-2 (GRK2), a serine-threonine protein kinase that induces internalization of chemokine receptors. Finally, we find that individuals who did not recover from sepsis had significantly more soluble ST2 (sST2, the decoy receptor of IL-33) than those who did recover. Together, our results indicate a previously undescribed mechanism of action of IL-33 and suggest a therapeutic potential of IL-33 in sepsis.
Aim
To evaluate whether Porphyromonas gingivalis‐induced periodontitis aggravates the antigen‐induced arthritis (AIA) model, and whether this effect is dependent on the Th17/IL‐17 signalling pathway.
...Materials and methods
Antigen‐induced arthritis was triggered by local injection of methylated bovine serum albumin into the knee joint of previously immunized C57BL/6 wild‐type (WT) and IL‐17 receptor A (IL‐17RA)‐knockout mice. Periodontal disease in naïve or arthritic mice was induced by oral infection with P. gingivalis. Animals were sacrificed 7, 15 and 30 days after infection. Alveolar bone loss, joint histopathology, articular hyperalgesia and joint cytokine production were assessed, in addition to the proportion of Th17 and Treg cells isolated from the inguinal lymph nodes.
Results
No influence of experimentally‐induced arthritis was found on the alveolar bone resorption induced by P. gingivalis. However, mice with experimentally‐induced arthritis that were exposed to P. gingivalis presented higher joint damage and Th17 frequencies when compared to non‐infected mice. The aggravation of arthritis by periodontitis was accompanied by increased TNF and IL‐17 production and articular neutrophil infiltration, whereas arthritis aggravation and changes in neutrophil infiltration were absent in IL‐17RA‐deficient mice.
Conclusion
The effects of P. gingivalis‐induced periodontitis on arthritis are dependent on Th17 expansion and IL‐17RA signalling, which lead to increased neutrophil infiltration into the joints.
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