Complement Receptor Research Articles

Molecular mechanism of complement inhibition by the trypanosome receptor ISG65.

African trypanosomes replicate within infected mammals where they are exposed to the complement system. This system centres around complement C3, which is present in a soluble form in serum but becomes covalently deposited onto the surfaces of pathogens after proteolytic cleavage to C3b. Membrane-associated C3b triggers different complement-mediated effectors which promote pathogen clearance. To counter complement-mediated clearance, African trypanosomes have a cell surface receptor, ISG65, which binds to C3b and which decreases the rate of trypanosome clearance in an infection model. However, the mechanism by which ISG65 reduces C3b function has not been determined. We reveal through cryogenic electron microscopy that ISG65 has two distinct binding sites for C3b, only one of which is available in C3 and C3d. We show that ISG65 does not block the formation of C3b or the function of the C3 convertase which catalyses the surface deposition of C3b. However, we show that ISG65 forms a specific conjugate with C3b, perhaps acting as a decoy. ISG65 also occludes the binding sites for complement receptors 2 and 3, which may disrupt recruitment of immune cells, including B cells, phagocytes, and granulocytes. This suggests that ISG65 protects trypanosomes by combining multiple approaches to dampen the complement cascade.

Current research on the interaction between Helicobacter pylori and macrophages.

Helicobacter pylori (H. pylori) is a gram-negative bacteria with a worldwide infection rate of 50%, known to induce gastritis, ulcers and gastric cancer. The interplay between H. pylori and immune cells within the gastric mucosa is pivotal in the pathogenesis of H. pylori-related disease. Following H. pylori infection, there is an observed increase in gastric mucosal macrophages, which are associated with the progression of gastritis. H. pylori elicits macrophage polarization, releases cytokines, reactive oxygen species (ROS) and nitric oxide (NO) to promote inflammatory response and eliminate H. pylori. Meanwhile, H. pylori has developed mechanisms to evade the host immune response in order to maintain the persistent infection, including interference with macrophage phagocytosis and antigen presentation, as well as induction of macrophage apoptosis. Consequently, the interaction between H. pylori and macrophages can significantly impact the progression, pathogenesis, and resolution of H. pylori infection. Moreover, macrophages are emerging as potential therapeutic targets for H. pylori-associated gastritis. Therefore, elucidating the involvement of macrophages in H. pylori infection may provide novel insights into the pathogenesis, progression, and management of H. pylori-related disease.

Macrophage-enriched novel functional long noncoding RNAs LRRC75A-AS1 and GAPLINC regulate polarization and innate immune responses.

Macrophages (Mφs) are functionally dynamic immune cells that bridge innate and adaptive immune responses; however, the underlying epigenetic mechanisms that control Mφ plasticity and innate immune functions are not well elucidated. To identify novel functions of macrophage-enriched lncRNAs in regulating polarization and innate immune responses. Total RNA isolated from differentiating monocyte-derived M1 and M2 Mφs was profiled for lncRNAs expression using RNAseq. Impact of LRRC75A-AS1, GAPLINC and AL139099.5 knockdown was examined on macrophage differentiation,polarization markers, phagocytosis, and antigen processing by flow cytometry and florescence microscopy. Cytokine profiles were examined by multiplex bead array and cytoskeletal signaling pathway genes were quantified by PCR-based array. Gingival biopsies were collected from periodontally healthy and diseased subjects to examine lncRNAs,M1/M2marker expression. Transcriptome profiling of M1 and M2 Mφs identified thousands of differentially expressed known and novel lncRNAs. We characterized three Mφ-enriched lncRNAs LRRC75A-AS1, GAPLINC and AL139099.5 in polarization and innate immunity. Knockdown of LRRC75A-AS1 and GAPLINC downregulated the Mφ differentiation markers and skewed Mφ polarization by decreasing M1 markers without a significant impact on M2 markers. LRRC75A-AS1 and GAPLINC knockdown also attenuated bacterial phagocytosis, antigen processing and inflammatory cytokine secretion in Mφs, supporting their functional role in potentiating innate immune functions. Mechanistically, LRRC75A-AS1 and GAPLINC knockdown impaired Mφ migration by downregulating the expression of multiple cytoskeletal signaling pathways suggesting their critical role in regulating Mφ migration. Finally, we showed that LRRC75A-AS1 and GAPLINC were upregulated in periodontitis and their expression correlates with higher M1 markers suggesting their role in macrophage polarizationin vivo. Our results show that polarized Mφs acquire a unique lncRNA repertoire and identified many previously unknown lncRNA sequences. LRRC75A-AS1 and GAPLINC, which are induced in periodontitis, regulate Mφ polarization and innate immune functions supporting their critical role in inflammation.

CSMD1 regulates brain complement activity and circuit development

Complement proteins facilitate synaptic elimination during neurodevelopmental pruning, but neural complement regulation is not well understood. CUB and Sushi Multiple Domains 1 (CSMD1) can regulate complement activity in vitro, is expressed in the brain, and is associated with increased schizophrenia risk. Beyond this, little is known about CSMD1 including whether it regulates complement activity in the brain or otherwise plays a role in neurodevelopment. We used biochemical, immunohistochemical, and proteomic techniques to examine the regional, cellular, and subcellular distribution as well as protein interactions of CSMD1 in the brain. To evaluate whether CSMD1 is involved in complement-mediated synapse elimination, we examined Csmd1-knockout mice and CSMD1-knockout human stem cell-derived neurons. We interrogated synapse and circuit development of the mouse visual thalamus, a process that involves complement pathway activity. We also quantified complement deposition on synapses in mouse visual thalamus and on cultured human neurons. Finally, we assessed uptake of synaptosomes by cultured microglia. We found that CSMD1 is present at synapses and interacts with complement proteins in the brain. Mice lacking Csmd1 displayed increased levels of complement component C3, an increased colocalization of C3 with presynaptic terminals, fewer retinogeniculate synapses, and aberrant segregation of eye-specific retinal inputs to the visual thalamus during the critical period of complement-dependent refinement of this circuit. Loss of CSMD1 in vivo enhanced synaptosome engulfment by microglia in vitro, and this effect was dependent on activity of the microglial complement receptor, CR3. Finally, human stem cell-derived neurons lacking CSMD1 were more vulnerable to complement deposition. These data suggest that CSMD1 can function as a regulator of complement-mediated synapse elimination in the brain during development.

Abstract 725: Listeria-inspired phagosome escape drives STING responses to CD47 blockade

Abstract Purpose: Through phagocytosis, antigen-presenting cells (APCs) engulf and neutralize malignant cells. ​However, engulfed tumor cells are destroyed in phagolysosomes leaving few peptides to enter cytosol, where cross-presentation of antigens and immune activation occurs. Listeria (L.) monocytogenes escape phagolysosomes by secreting a pore-forming protein Listeriolysin O (LLO). Our breakthrough centers on harnessing LLO to create LLO-CD47, a novel protein-antibody conjugate that fuses the myeloid checkpoint anti-CD47 to LLO. By promoting tumor cell-phagocytosis and the release of tumor peptides and DNA from phagolysosomes, we hypothesize that LLO-CD47 enhances the immune functionalities of APCs. Procedures: Anti-CD47 was conjugated to LLO using a water-soluble SPDP crosslinker and purified by affinity chromatography. Transmission electron microscopy (TEM) was used to visualize the integrity of macrophage phagolysosomes following treatment. C57BL/6 mouse bone marrow-derived macrophages (BMDMs) were used to study M2-to-Ml polarization, tumor cell phagocytosis, STING activation, and antigen presentation. Mice bearing orthotopically implanted spontaneously metastatic murine triple-negative 4T1 tumors and E0771 tumors received intratumoral (IT) or intraperitoneal (IP) injection. CD8+ T cells or tumor-associated macrophages (TAMs) were depleted from tumor-bearing mice using an anti-CD8 antibody or anti-CSF-1R antibody prior to treatment. Results: 1. LLO-CD47 skews BMDMs from M2-to-M1 inflammatory phenotypes and enhances the phagocytosis of E0771 tumor cells. 2. BMDMs visualized by TEM show breaches in phagosome membranes following LLO-CD47, but not anti-CD47, treatment. 3. LLO-CD47 increases levels of phosphorylated STING, IFN, and TNFa in BMDMs in vitro and TAMs in vivo, relative to anti-CD47. 4. IT delivery of LLO-CD47 inhibits the growth and metastasis of orthotopically implanted murine breast tumors relative to anti-CD47. Harvested tumors show increased M1 polarized macrophages and CD8+ T cells, which express high levels of PD-1. 5. Co-administration of IP anti-PD-1 and LLO-CD47 delays the progression of breast tumor metastasis and prolongs animal survival relative to animals treated with anti-PD-1 and anti-CD47. 6. The elimination of CD8+ T cells or TAMs abrogates the antitumor effect of LLO-CD47. 7. IP administration of LLO-CD47 showed acceptable toxicity with stable body weight gain and no long-term hematological, renal, or hepatic toxicities. Conclusion: Conjugation of LLO to anti-CD47 enhances macrophage STING activation, antigen presentation, and tumor cell phagocytosis. Our work represents the first antibody-drug conjugate specifically designed to enhance immune activation against cancer.​ Citation Format: Benjamin R. Schrank, Yifan Wang, Abin Antony, Wen Jiang. Listeria-inspired phagosome escape drives STING responses to CD47 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 725.

Restoring SRSF3 in Kupffer cells attenuates obesity-related insulin resistance.

In obesity, depletion of KCs expressing CRIg (complement receptor of the Ig superfamily) leads to microbial DNA accumulation, which subsequently triggers tissue inflammation and insulin resistance. However, the mechanism underlying obesity-mediated changes in KC complement immune functions is largely unknown. Using KC-specific deactivated Cas9 transgenic mice treated with guide RNA, we assessed the effects of restoring CRIg or the serine/arginine-rich splicing factor 3 (SRSF3) abundance on KC functions and metabolic phenotypes in obese mice. The impacts of weight loss on KC responses were evaluated in a diet switch mouse model. The role of SRSF3 in regulating KC functions was also evaluated using KC-specific SRSF3 knockout mice. Here, we report that overexpression of CRIg in KCs of obese mice protects against bacterial DNA accumulation in metabolic tissues. Mechanistically, SRSF3 regulates CRIg expression, which is essential for maintaining the CRIg+ KC population. During obesity, SRSF3 expression decreases, but it is restored with weight loss through a diet switch, normalizing CRIg+ KCs. KC SRSF3 is also repressed in obese human livers. Lack of SRSF3 in KCs in lean and obese mice decreases their CRIg+ population, impairing metabolic parameters. During the diet switch, the benefits of weight loss are compromised due to SRSF3 deficiency. Conversely, SRSF3 overexpression in obese mice preserves CRIg+ KCs and improves metabolic responses. Restoring SRSF3 abundance in KCs offers a strategy against obesity-associated tissue inflammation and insulin resistance by preventing bacterial DNA accumulation.

Dantu Blood Group Erythrocytes Form Large Plasmodium falciparum Rosettes Less Commonly.

Dantu erythrocytes, which express a hybrid glycophorin B/A protein, are protective against severe malaria. Recent studies have shown that Dantu impairs Plasmodium falciparum invasion by increasing erythrocyte membrane tension, but its effects on pathological host-parasite adhesion interactions such as rosetting, the binding of uninfected erythrocytes to P. falciparum-infected erythrocytes, have not been investigated previously. The expression of several putative host rosetting receptors-including glycophorin A (GYPA), glycophorin C (GYPC), complement receptor 1 (CR1), and band 3, which complexes with GYPA to form the Wrightb blood group antigen-are altered on Dantu erythrocytes. Here, we compare receptor expression, and rosetting at both 1 hour and 48 hours after mixing with mature trophozoite-stage Kenyan laboratory-adapted P. falciparum strain 11019 parasites in Dantu and non-Dantu erythrocytes. Dantu erythrocytes showed lower staining for GYPA and CR1, and greater staining for band 3, as observed previously, whereas Wrightb and GYPC staining did not vary significantly. No significant between-genotype differences in rosetting were seen after 1 hour, but the percentage of large rosettes was significantly less in both Dantu heterozygous (mean, 16.4%; standard error of the mean [SEM], 3.2) and homozygous donors (mean, 15.4%; SEM, 1.4) compared with non-Dantu erythrocytes (mean, 32.9%; SEM, 7.1; one-way analysis of variance, P = 0.025) after 48 hours. We also found positive correlations between erythrocyte mean corpuscular volume (MCV), the percentage of large rosettes (Spearman's rs = 0.5970, P = 0.0043), and mean rosette size (rs = 0.5206, P = 0.0155). Impaired rosetting resulting from altered erythrocyte membrane receptor expression and reduced MCV might add to the protective effect of Dantu against severe malaria.

Neutrophil-inflicted vasculature damage suppresses immune-mediated optic nerve regeneration

In adult mammals, injured retinal ganglion cells (RGCs) fail to spontaneously regrow severed axons, resulting in permanent visual deficits. Robust axon growth, however, is observed after intra-ocular injection of particulate β-glucan isolated from yeast. Blood-borne myeloid cells rapidly respond to β-glucan, releasing numerous pro-regenerative factors. Unfortunately, the pro-regenerative effects are undermined by retinal damage inflicted by an overactive immune system. Here, we demonstrate that protection of the inflamed vasculature promotes immune-mediated RGC regeneration. In the absence of microglia, leakiness of the blood-retina barrier increases, pro-inflammatory neutrophils are elevated, and RGC regeneration is reduced. Functional ablation of the complement receptor 3 (CD11b/integrin-αM), but not the complement components C1q-/- or C3-/-, reduces ocular inflammation, protects the blood-retina barrier, and enhances RGC regeneration. Selective targeting of neutrophils with anti-Ly6G does not increase axogenic neutrophils but protects the blood-retina barrier and enhances RGC regeneration. Together, these findings reveal that protection of the inflamed vasculature promotes neuronal regeneration.

Control of complement-induced inflammatory responses to SARS-CoV-2 infection by anti-SARS-CoV-2 antibodies.

Dysregulated immune responses contribute to the excessive and uncontrolled inflammation observed in severe COVID-19. However, how immunity to SARS-CoV-2 is induced and regulated remains unclear. Here, we uncover the role of the complement system in the induction of innate and adaptive immunity to SARS-CoV-2. Complement rapidly opsonizes SARS-CoV-2 particles via the lectin pathway. Complement-opsonized SARS-CoV-2 efficiently induces type-I interferon and pro-inflammatory cytokine responses via activation of dendritic cells, which are inhibited by antibodies against the complement receptors (CR) 3 and 4. Serum from COVID-19 patients, or monoclonal antibodies against SARS-CoV-2, attenuate innate and adaptive immunity induced by complement-opsonized SARS-CoV-2. Blocking of CD32, the FcγRII antibody receptor of dendritic cells, restores complement-induced immunity. These results suggest that opsonization of SARS-CoV-2 by complement is involved in the induction of innate and adaptive immunity to SARS-CoV-2 in the acute phase of infection. Subsequent antibody responses limit inflammation and restore immune homeostasis. These findings suggest that dysregulation of the complement system and FcγRII signaling may contribute to severe COVID-19.

Triple-fusion protein (TriFu): A potent, targeted, enzyme-like inhibitor of all three complement activation pathways

The introduction of a therapeutic anti-C5 antibody into clinical practice in 2007 inspired a surge into the development of complement-targeted therapies. This has led to the recent approval of a C3 inhibitory peptide, an antibody directed against C1s and a full pipeline of several complement inhibitors in preclinical and clinical development. However, no inhibitor is available that efficiently inhibits all three complement initiation pathways and targets host cell surface markers as well as complement opsonins. To overcome this, we engineered a novel fusion protein combining selected domains of the three natural complement regulatory proteins decay accelerating factor, factor H and complement receptor 1. Such a triple fusion complement inhibitor (TriFu) was recombinantly expressed and purified alongside multiple variants and its building blocks. We analyzed these proteins for ligand binding affinity and decay acceleration activity by surface plasmon resonance. Additionally, we tested complement inhibition in several invitro/exvivo assays using standard classical and alternative pathway restricted hemolysis assays next to hemolysis assays with paroxysmal nocturnal hemoglobinuria erythrocytes. A novel invitro model of the alternative pathway disease C3 glomerulopathy was established to evaluate the potential of the inhibitors to stop C3 deposition on endothelial cells. Next to the novel engineered triple fusion variants which inactivate complement convertases in an enzyme-like fashion, stoichiometric complement inhibitors targeting C3, C5, factor B, and factor D were tested as comparators. The triple fusion approach yielded a potent complement inhibitor that efficiently inhibits all three complement initiation pathways while targeting to surface markers.

Macrophage-1 antigen exacerbates histone-induced acute lung injury and promotes neutrophil extracellular trap formation.

Acute lung injury (ALI), which occurs in association with sepsis, trauma, and coronavirus disease 2019 (COVID-19), is a serious clinical condition with high mortality. Excessive platelet-leukocyte aggregate (PLA) formation promotes neutrophil extracellular trap (NET) release and thrombosis, which are involved in various diseases, including ALI. Macrophage-1 antigen (Mac-1, CD11b/CD18), which is expressed on the surface of leukocytes, is known to promote NET formation. This study aimed to elucidate the role of Mac-1 in extracellular histone-induced ALI. Exogenous histones were administered to Mac-1-deficient mice and wild-type (WT) mice with or without neutrophil or platelet depletion, and several parameters were investigated 1 h after histone injection. Depletion of neutrophils or platelets improved survival time and macroscopic and microscopic properties of lung tissues, and decreased platelet-leukocyte formation and plasma myeloperoxidase levels. These improvements were also observed in Mac-1-/- mice. NET formation in Mac-1-/- bone marrow neutrophils (BMNs) was significantly lower than that in WT BMNs. In conclusion, our findings suggest that Mac-1 is associated with exacerbation of histone-induced ALI and the promotion of NET formation in the presence of activated platelets.

Amyloid-β aggregates activate peripheral monocytes in mild cognitive impairment

The peripheral immune system is important in neurodegenerative diseases, both in protecting and inflaming the brain, but the underlying mechanisms remain elusive. Alzheimer’s Disease is commonly preceded by a prodromal period. Here, we report the presence of large Aβ aggregates in plasma from patients with mild cognitive impairment (n = 38). The aggregates are associated with low level Alzheimer’s Disease-like brain pathology as observed by 11C-PiB PET and 18F-FTP PET and lowered CD18-rich monocytes. We characterize complement receptor 4 as a strong binder of amyloids and show Aβ aggregates are preferentially phagocytosed and stimulate lysosomal activity through this receptor in stem cell-derived microglia. KIM127 integrin activation in monocytes promotes size selective phagocytosis of Aβ. Hydrodynamic calculations suggest Aβ aggregates associate with vessel walls of the cortical capillaries. In turn, we hypothesize aggregates may provide an adhesion substrate for recruiting CD18-rich monocytes into the cortex. Our results support a role for complement receptor 4 in regulating amyloid homeostasis.

Increased expression of complement C3c, iC3b, and cells containing CD11b or CD14 in experimentally induced psoriatic lesion.

Psoriasis is a chronic inflammatory skin disease with a characteristic isomorphic reaction, i.e. the Köbner reaction, induced by slight epidermal trauma. In this study, the tape-stripping technique was used to induce the development of Köbner reaction in 18 subjects with psoriasis. Eight subjects developed a positive reaction. To study the early cellular changes, skin biopsies were taken at the baseline and subsequent time points of 2 h, 1 d, 3 d, and 7 d for the immunostaining of complement C3c, iC3b, and cells expressing complement receptor 3 (CD11b/CD18; a receptor of iC3b) or CD14. The results show that the positive Köbner reaction is associated with rapid (2 h-1 d) and sustained (3-7 d) increase in the expression of epidermal C3c and iC3b and dermal C3c. In addition, there was a positive correlation between CD11b+ and CD14+ cells in baseline and 2 h-1 d biopsies with a subsequent increase in CD11b+ and CD14+ cells in 3-7 d biopsies in the Köbner-positive group. In the Köbner-negative group, only a transient increase in epidermal iC3b at 2 h-1 d, as well as rapid (2 h-1 d) and sustained increase (3-7 d) in dermal iC3b and CD14+ cells, was observed. In experiments with cultured monolayer keratinocytes, a slight cell damage already at 30 mJ/cm2 ultraviolet B irradiation led to increased expression of C3c, but not iC3b. Therefore, there are marked differences between Köbner groups in respect to the expression of C3c, iC3b, and cells expressing CD11b or CD14. Of note is the rapid and sustained increase in epidermal C3c and iC3b in the positive Köbner reaction.

Complement receptor 3 is required for maximum in vitro trogocytic killing of the parasite Trichomonas vaginalis by human neutrophil-like cells.

Trichomonas vaginalis (Tv) is a parasite that causes trichomoniasis, a prevalent sexually-transmitted infection. Neutrophils are found at the site of infection, and can rapidly kill the parasite in vitro, using trogocytosis. However, the specific molecular players in neutrophil killing of Tv are unknown. Here, we show that complement proteins play a role in Tv killing by human neutrophil-like cells (NLCs). Using CRISPR/Cas9, we generated NLCs deficient in each of three complement receptors (CRs) known to be expressed on human neutrophils: CR1, CR3, and CR4. Using in vitro trogocytosis assays, we found that CR3, but not CR1 or CR4 is required for maximum trogocytosis of the parasite by NLCs, with NLCs lacking CR3 demonstrating ~40% reduction in trogocytosis, on average. We also observed a reduction in NLC killing of Tv in CR3 knockout, but not CR1 or CR4 knockout NLCs. On average, NLCs lacking CR3 had ~50% reduction in killing activity. We also used a parallel approach of pre-incubating NLCs with blocking antibodies against CR3, which similarly reduced NLC killing of parasites. These data support a model in which Tv is opsonized by the complement protein iC3b, and bound by neutrophil CR3 receptor, to facilitate trogocytic killing of the parasite.

Activation of leukotriene B4 receptor 1 is a prerequisite for complement receptor 3-mediated antifungal responses of neutrophils.

Invasive fungal infections are life-threatening, and neutrophils are vital cells of the innate immune system that defend against them. The role of LTA4H-LTB4-BLT1 axis in regulation of neutrophil responses to fungal infection remains poorly understood. Here, we demonstrated that the LTA4H-LTB4-BLT1 axis protects the host against Candida albicans and Aspergillus fumigatus, but not Cryptococcus neoformans infection, by regulating the antifungal activity of neutrophils. Our results show that deleting Lta4h or Blt1 substantially impairs the fungal-specific phagocytic capacity of neutrophils. Moreover, defective activation of the spleen tyrosine kinase (Syk) and extracellular signal-related kinase (ERK1/2) pathways in neutrophils accompanies this impairment. Mechanistically, BLT1 regulates CR3-mediated, β-1,3-glucan-induced neutrophil phagocytosis, while a physical interaction with CR3 with slight influence on its dynamics is observed. Our findings thus demonstrate that the LTA4H-LTB4-BLT1 axis is essential for the phagocytic function of neutrophils in host antifungal immune response against Candida albicans and Aspergillus fumigatus.

Mac-1 deficiency ameliorates pressure overloaded heart failure through inhibiting macrophage polarization and activation

Persistent pressure overload commonly leads to pathological cardiac hypertrophy and remodeling, ultimately leading to heart failure (HF). Cardiac remodeling is associated with the involvement of immune cells and the inflammatory response in pathogenesis. The macrophage-1 antigen (Mac-1) is specifically expressed on leukocytes and regulates their migration and polarization. Nonetheless, the involvement of Mac-1 in cardiac remodeling and HF caused by pressure overload has not been determined. The Mac-1-knockout (KO) and wild-type (WT) mice were subjected to transverse aortic constriction (TAC) for 6 weeks. Echocardiography and pressure–volume loop assessments were used to evaluate cardiac function, and cardiac remodeling and macrophage infiltration and polarization were estimated by histopathology and molecular techniques. The findings of our study demonstrated that Mac-1 expression was markedly increased in hearts subjected to TAC treatment. Moreover, compared with WT mice, Mac-1-KO mice exhibited dramatically ameliorated TAC-induced cardiac dysfunction, hypertrophy, fibrosis, oxidative stress and apoptosis. The potential positive impacts may be linked to the inhibition of macrophage infiltration and M1 polarization via reductions in NF-kB and STAT1 expression and upregulation of STAT6. In conclusion, this research reveals a new function of Mac-1 deficiency in reducing pathological cardiac remodeling and HF caused by pressure overload. Additionally, inhibiting Mac-1 could be a potential treatment option for patients with HF in a clinical setting.

Preclinical evaluation of the gorilla-derived HAdV-B AdV-lumc007 oncolytic adenovirus 'GoraVir' for the treatment of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy which shows unparalleled therapeutic resistance due to its genetic and cellular heterogeneity, dense stromal tissue, and immune-suppressive tumour microenvironment. Oncolytic virotherapy has emerged as a new treatment modality which uses tumour-specific viruses to eliminate cancerous cells. Non-human primate adenoviruses of the human adenovirus B (HAdV-B) species have demonstrated considerable lytic potential in humancancer cells as well as limited preexisting neutralizing immunity in humans. Previously, we have generated a new oncolytic derivative of the gorilla-derived HAdV-B AdV-lumc007 named 'GoraVir'. Here, we show that GoraVir displays oncolytic efficacy in pancreatic cancer cells and pancreatic-cancer-associated fibroblasts. Moreover, it retains its lytic potential in monoculture and co-culture spheroids. In addition, we established the ubiquitously expressed complement receptor CD46 as the main entry receptor for GoraVir. Finally, a single intratumoural dose of GoraVir was shown to delay tumour growth in a BxPC-3 xenograft model at 10 days post-treatment. Collectively, these data demonstrate that the new gorilla-derived oncolytic adenovirus is a potent oncolytic vector candidate that targets both pancreatic cancer cells and tumour-adjacent stroma.

Complement receptor 1 is the human erythrocyte receptor for Plasmodium vivax erythrocyte binding protein

The discovery that Africans were resistant to infection by Plasmodium vivax (P. vivax) led to the conclusion that P. vivax invasion relied on the P. vivax Duffy Binding Protein (PvDBP) interacting with the Duffy Antigen Receptor for Chemokines (DARC) expressed on erythrocytes. However, the recent reporting of P. vivax infections in DARC-negative Africans suggests that the parasite might use an alternate invasion pathway to infect DARC-negative reticulocytes. To identify the parasite ligands and erythrocyte receptors that enable P. vivax invasion of both DARC-positive and -negative erythrocytes, we expressed region II containing the Duffy Binding-Like (DBL) domain of P. vivax erythrocyte binding protein (PvEBP-RII) and verified that the DBL domain binds to both DARC-positive and -negative erythrocytes. Furthermore, an AVidity-based EXtracelluar Interaction Screening (AVEXIS) was used to identify the receptor for PvEBP among over 750 human cell surface receptor proteins, and this approach identified only Complement Receptor 1 (CR1, CD35, or C3b/C4b receptor) as a PvEBP receptor. CR1 is a well-known receptor for P. falciparum Reticulocyte binding protein Homology 4 (PfRh4) and is present on the surfaces of both reticulocytes and normocytes, but its expression decreases as erythrocytes age. Indeed, PvEBP-RII bound to a subpopulation of both reticulocytes and normocytes, and this binding was blocked by the addition of soluble CR1 recombinant protein, indicating that CR1 is the receptor of PvEBP. In addition, we found that the Long Homology Repeat A (LHR-A) subdomain of CR1 is the only subdomain responsible for mediating the interaction with PvEBP-RII.

Salmonella manipulates the host to drive pathogenicity via induction of interleukin 1β.

Acute gastrointestinal infection with intracellular pathogens like Salmonella Typhimurium triggers the release of the proinflammatory cytokine interleukin 1β (IL-1β). However, the role of IL-1β in intestinal defense against Salmonella remains unclear. Here, we show that IL-1β production is detrimental during Salmonella infection. Mice lacking IL-1β (IL-1β -/-) failed to recruit neutrophils to the gut during infection, which reduced tissue damage and prevented depletion of short-chain fatty acid (SCFA)-producing commensals. Changes in epithelial cell metabolism that typically support pathogen expansion, such as switching energy production from fatty acid oxidation to fermentation, were absent in infected IL-1β -/- mice which inhibited Salmonella expansion. Additionally, we found that IL-1β induces expression of complement anaphylatoxins and suppresses the complement-inactivator carboxypeptidase N (CPN1). Disrupting this process via IL-1β loss prevented mortality in Salmonella-infected IL-1β -/- mice. Finally, we found that IL-1β expression correlates with expression of the complement receptor in patients suffering from sepsis, but not uninfected patients and healthy individuals. Thus, Salmonella exploits IL-1β signaling to outcompete commensal microbes and establish gut colonization. Moreover, our findings identify the intersection of IL-1β signaling and the complement system as key host factors involved in controlling mortality during invasive Salmonellosis.

Conformational variants of the ternary complex of C5a, C5aR1, and G-protein

The complement component fragment 5a (C5a) binds and activates two complement receptors like C5aR1 and C5aR2, which play a significant role in orchestrating the proinflammatory function of C5a in tissues through the recruitment of heterotrimeric G-proteins and β-arrestins. Dysregulation of the complement induces excessive production of C5a, which triggers aberrant activation of the C5a-C5aR1-G-protein and C5a-C5aR2-β-arrestin signalling axes in tissues, contributing to the pathology of numerous immune-inflammatory diseases. Thus, understanding the interaction of C5a with C5aR1 and C5aR2, as well as the interaction of G-protein and β-arrestins, respectively, with C5a-C5aR1 and C5a-C5aR2, holds tremendous therapeutic value. In the absence of structural data, we have previously elaborated the binary complexes of C5a-C5aR1 and C5a-C5aR2, as well as the ternary complex of C5a-C5aR2-β-arrestin1, in highly refined model structures. While our ternary model complex of C5a-C5aR1-G-protein was in progress, two cryo-electron microscopy-based ternary structural complexes of C5aR1 were made available by others. However, it is observed that the interaction of the crucial NT-peptide of C5aR1 with C5a, including the portion of the G⍺i-subunit that harbors the switch-I region, is not fully resolved in both complexes. The current study addresses the issues and provides two highly refined alternative model ternary complexes of C5a-C5aR1-G-protein. The study highlights the conformational heterogeneity in C5aR1 by comparing the two conformational variants of the model ternary complex in the context of C5a-C5aR2-β-arrestin1 for further devising methods and molecules targeting both surface and intracellular C5aR1/C5aR2 for effectively mitigating the proinflammatory role of C5a in various disease settings. Communicated by Ramaswamy H. Sarma