C3 Activation Research Articles

Complement C3 deposition restricts the proliferation of internalized Staphylococcus aureus by promoting autophagy.

Complement C3 (C3) is usually deposited spontaneously on the surfaces of invading bacteria prior to internalization, but the impact of C3 coating on cellular responses is largely unknown. Staphylococcus aureus (S. aureus) is a facultative intracellular pathogen that subverts autophagy and replicates in both phagocytic and nonphagocytic cells. In the present study, we deposited C3 components on the surface of S. aureus by complement opsonization before cell infection and confirmed that C3-coatings remained on the surface of the bacteria after they have invaded the cells, suggesting S. aureus cannot escape or degrade C3 labeling. We found that the C3 deposition on S. aureus notably enhanced cellular autophagic responses, and distinguished these responses as xenophagy, in contrast to LC3-associated phagocytosis (LAP). Furthermore, this upregulation was due to the recruitment of and direct interaction with autophagy-related 16-like 1 (ATG16L1), thereby resulting in autophagy-dependent resistance to bacterial growth within cells. Interestingly, this autophagic effect occurred only after C3 activation by enzymatic cleavage because full-length C3 without cleavage of the complement cascade reaction, although capable of binding to ATG16L1, failed to promote autophagy. These findings demonstrate the biological function of intracellular C3 upon bacterial infection in enhancing autophagy against internalized S. aureus.

Inhibition of the C1s Protease and the Classical Complement Pathway by 6-(4-Phenylpiperazin-1-yl)Pyridine-3-Carboximidamide and Chemical Analogs.

The classical pathway (CP) is a potent mechanism for initiating complement activity and is a driver of pathology in many complement-mediated diseases. The CP is initiated via activation of complement component C1, which consists of the pattern recognition molecule C1q bound to a tetrameric assembly of proteases C1r and C1s. Enzymatically active C1s provides the catalytic basis for cleavage of the downstream CP components, C4 and C2, and is therefore an attractive target for therapeutic intervention in CP-driven diseases. Although an anti-C1s mAb has been Food and Drug Administration approved, identifying small-molecule C1s inhibitors remains a priority. In this study, we describe 6-(4-phenylpiperazin-1-yl)pyridine-3-carboximidamide (A1) as a selective, competitive inhibitor of C1s. A1 was identified through a virtual screen for small molecules that interact with the C1s substrate recognition site. Subsequent functional studies revealed that A1 dose-dependently inhibits CP activation by heparin-induced immune complexes, CP-driven lysis of Ab-sensitized sheep erythrocytes, CP activation in a pathway-specific ELISA, and cleavage of C2 by C1s. Biochemical experiments demonstrated that A1 binds directly to C1s with a Kd of ∼9.8 μM and competitively inhibits its activity with an inhibition constant (Ki) of ∼5.8 μM. A 1.8-Å-resolution crystal structure revealed the physical basis for C1s inhibition by A1 and provided information on the structure-activity relationship of the A1 scaffold, which was supported by evaluating a panel of A1 analogs. Taken together, our work identifies A1 as a new class of small-molecule C1s inhibitor and lays the foundation for development of increasingly potent and selective A1 analogs for both research and therapeutic purposes.

Liver macrophages and sinusoidal endothelial cells execute vaccine-elicited capture of invasive bacteria.

Vaccination has substantially reduced the morbidity and mortality of bacterial diseases, but mechanisms of vaccine-elicited pathogen clearance remain largely undefined. We report that vaccine-elicited immunity against invasive bacteria mainly operates in the liver. In contrast to the current paradigm that migrating phagocytes execute vaccine-elicited immunity against blood-borne pathogens, we found that invasive bacteria are captured and killed in the liver of vaccinated host via various immune mechanisms that depend on the protective potency of the vaccine. Vaccines with relatively lower degrees of protection only activated liver-resident macrophage Kupffer cells (KCs) by inducing pathogen-binding immunoglobulin M (IgM) or low amounts of IgG. IgG-coated pathogens were directly captured by KCs via multiple IgG receptors FcγRs, whereas IgM-opsonized bacteria were indirectly bound to KCs via complement receptors of immunoglobulin superfamily (CRIg) and complement receptor 3 (CR3) after complement C3 activation at the bacterial surface. Conversely, the more potent vaccines engaged both KCs and liver sinusoidal endothelial cells by inducing higher titers of functional IgG antibodies. Endothelial cells (ECs) captured densely IgG-opsonized pathogens by the low-affinity IgG receptor FcγRIIB in a "zipper-like" manner and achieved bacterial killing predominantly in the extracellular milieu via an undefined mechanism. KC- and endothelial cell-based capture of antibody-opsonized bacteria also occurred in FcγR-humanized mice. These vaccine protection mechanisms in the liver not only provide a comprehensive explanation for vaccine-/antibody-boosted immunity against invasive bacteria but also may serve as in vivo functional readouts of vaccine efficacy.

Loss of Complement Factor I (Cfi) a Negative Regulator of Complement Cascade Activity Exacerbates JAK2V617F-Dependent Phenotype

Background: The current evidence suggests that inflammatory phenotype contributes to the pathogenesis of myeloproliferative neoplasms (MPNs). While our understanding of complement cascade contributions to inflammation-mediated pathologies has expanded, role this cascade plays in etiology of myeloid malignancies has not been detailed. Previously, we uncovered an inactivating RNSV in complement factor I (CFI G119R), an inhibitor of the complement cascade, in 20% of PMF patients (n=10) using WGS, and in a separate patient cohort (n=10) we observed an increase in CH50 levels (n=3) and decrease in C3 levels (n=3) (Oakes et al., 2021, Blood, 138:1472).These findings prompted us to generate murine models allowing assessment of the Cfi loss-driven complement overactivity effects on hematopoiesis and development of MPNs. Methods: A conventional Cfi knockout (KO) mouse model was generated by targeting exon 2 of the Cfi gene using CRISPR/Cas9. Inactivation of one or two copies of Cfi was confirmed by genotyping and total loss (Cfi KO) or Cfi deficiency (Cfi HET) were confirmed in peripheral blood (PB) plasma. Complete blood counts (CBC) and the frequencies of granulocytes, B cells, plasmocytes, T cells, macrophages/monocytes, platelets, erythroid cells in the bone marrow were longitudinally assessed by flow cytometry for 40 weeks in Cfi WT, HET and KO mice. Cfi HET animals were crossed with JAK2V617F(fl/+);Mx1-cre(+) mice (obtained from crossing B6.Cg-Tg( Mx1- cre+ 1)Cgn/J; # 003556, JAX) and JAK2V617F expressing (B6N.129S6(SJL)- Jak2 tm1.2Ble/AmlyJ #031658, JAX) to generate Cfi(+/-)/JAK2V617F(fl/+);Tg(Mx1-cre(+/-) (CFI/JAK2) strain. Complete blood counts and survival were monitored for Cfi(+/+)/JAK2V617F(fl/+);Tg(Mx1-cre)(+/-) (CFI WT/JAK2 HET) Cfi(+/-)/JAK2V617F(fl/+);Tg(Mx1-cre)(+/-) (CFI HET/JAK2 HET) or Cfi(-/-)/JAK2V617F(fl/+); Tg(Mx1-cre)(+/-) (CFI KO/JAK2 HET) mice. Results:Cfi HET and KO mice were fertile and viable. Cohort of Cfi WT n=13, Het n=12, KO n=13 mice was subjected to analyses. Genotypes were confirmed via PCR and western blot. Flow cytometry demonstrated a copy loss dependent decrease in total T-cells (CD3+) and increase in mature resting B-cells (CD19+/B220+). CBC demonstrated a copy loss dependent decrease in both monocyte (p adj. KO vs. WT 0.002) and granulocyte percentage (p adj. KO vs. WT 0.003), and increase in lymphocyte percentage (p adj. KO vs. WT 0.001). In PB plasma we detected increased C3 activation in KO mice compared to WT and Het, demonstrated as decreased C3 alpha and presence of inactivated C3 due to increased proteolytical processing of C3 complex. Proteolytic cleavage of CFB compared to Cfi WT and Het was also noted in Cfi KO animals. A significant decrease in survival probability of CFI HET/JAK2 HET vs. CFI WT/JAK2 HET (p=0.03), CFI KO/JAK2 HET vs. CFI WT/JAK2 HET (p=0.003), and CFI HET/JAK2 HET vs. CFI KO/JAK2 HET (p=0.05) averaging 92 days for Cfi HET/JAK2 HET, 67 days for Cfi KO/JAK2 HET vs. 98 days for Cfi WT/JAK2 HET was noted (Fig. A). A granulocyte and monocyte percentage of CFI HET/JAK2 HET was increased as compared to CFI WT/JAK2 HET mice (Fig. B). Conclusions:An overactivation of complement cascade at the C3 level is noted in Cfi deficient animals, that is phenotypically linked to an increase in frequency of B cells. These findings may suggest involvement of Cfi directly, and/or in conjunction with generally overactive complement, in B-cell maturation and function. The absence of one copy of Cfi in JAK2V617F mice shows more severe MPN disease progression resulting in markedly decreased survival, linked to increased frequencies of monocytes and granulocytes and markedly decreased lymphocytes. In sum, these data indicate that increased complement activity may increase JAK2V617F-dependent phenotype severity. In depth mechanistic studies detailing observed phenotype are warranted.

Mice deficient in complement C3 are protected against recognition memory deficits and astrogliosis induced by status epilepticus.

Status epilepticus (SE) can significantly increase the risk of temporal lobe epilepsy (TLE) and cognitive comorbidities. A potential candidate mechanism underlying memory defects in epilepsy may be the immune complement system. The complement cascade, part of the innate immune system, modulates inflammatory and phagocytosis signaling, and has been shown to contribute to learning and memory dysfunctions in neurodegenerative disorders. We previously reported that complement C3 is elevated in brain biopsies from human drug-resistant epilepsy and in experimental rodent models. We also found that SE-induced increases in hippocampal C3 levels paralleled the development of hippocampal-dependent spatial learning and memory deficits in rats. Thus, we hypothesized that SE-induced C3 activation contributes to this pathophysiology in a mouse model of SE and acquired TLE. In this study C3 knockout (KO) and wild type (WT) mice were subjected to one hour of pilocarpine-induced SE or sham conditions (control; C). Following a latent period of two weeks, recognition memory was assessed utilizing the novel object recognition (NOR) test. Western blotting was utilized to determine the protein levels of C3 in hippocampal lysates. In addition, we assessed the protein levels and distribution of the astrocyte marker glial fibrillary acidic protein (GFAP). In the NOR test, control WT + C or C3 KO + C mice spent significantly more time exploring the novel object compared to the familiar object. In contrast, WT+SE mice did not show preference for either object, indicating a memory defect. This deficit was prevented in C3 KO + SE mice, which performed similarly to controls. In addition, we found that SE triggered significant increases in the protein levels of GFAP in hippocampi of WT mice but not in C3 KO mice. These findings suggest that ablation of C3 prevents SE-induced recognition memory deficits and that a C3-astrocyte interplay may play a role. Therefore, it is possible that enhanced C3 signaling contributes to SE-associated cognitive decline during epileptogenesis and may serve as a potential therapeutic target for treating cognitive comorbidities in acquired TLE.

Efficacy and safety of baricitinib or ravulizumab in adult patients with severe COVID-19 (TACTIC-R): a randomised, parallel-arm, open-label, phase 4 trial

From early in the COVID-19 pandemic, evidence suggested a role for cytokine dysregulation and complement activation in severe disease. In the TACTIC-R trial, we evaluated the efficacy and safety of baricitinib, an inhibitor of Janus kinase 1 (JAK1) and JAK2, and ravulizumab, a monoclonal inhibitor of complement C5 activation, as an adjunct to standard of care for the treatment of adult patients hospitalised with COVID-19. TACTIC-R was a phase 4, randomised, parallel-arm, open-label platform trial that was undertaken in the UK with urgent public health designation to assess the potential of repurposing immunosuppressants for the treatment of severe COVID-19, stratified by a risk score. Adult participants (aged ≥18 years) were enrolled from 22 hospitals across the UK. Patients with a risk score indicating a 40% risk of admission to an intensive care unit or death were randomly assigned 1:1:1 to standard of care alone, standard of care with baricitinib, or standard of care with ravulizumab. The composite primary outcome was the time from randomisation to incidence (up to and including day 14) of the first event of death, invasive mechanical ventilation, extracorporeal membrane oxygenation, cardiovascular organ support, or renal failure. The primary interim analysis was triggered when 125 patient datasets were available up to day 14 in each study group and we included in the analysis all participants who were randomly assigned. The trial was registered on ClinicalTrials.gov (NCT04390464). Between May 8, 2020, and May 7, 2021, 417 participants were recruited and randomly assigned to standard of care alone (145 patients), baricitinib (137 patients), or ravulizumab (135 patients). Only 54 (39%) of 137 patients in the baricitinib group received the maximum 14-day course, whereas 132 (98%) of 135 patients in the ravulizumab group received the intended dose. The trial was stopped after the primary interim analysis on grounds of futility. The estimated hazard ratio (HR) for reaching the composite primary endpoint was 1·11 (95% CI 0·62-1·99) for patients on baricitinib compared with standard of care alone, and 1·53 (0·88-2·67) for ravulizumab compared with standard of care alone. 45 serious adverse events (21 deaths) were reported in the standard-of-care group, 57 (24 deaths) in the baricitinib group, and 60 (18 deaths) in the ravulizumab group. Neither baricitinib nor ravulizumab, as administered in this study, was effective in reducing disease severity in patients selected for severe COVID-19. Safety was similar between treatments and standard of care. The short period of dosing with baricitinib might explain the discrepancy between our findings and those of other trials. The therapeutic potential of targeting complement C5 activation product C5a, rather than the cleavage of C5, warrants further evaluation. UK Medical Research Council, UK National Institute for Health Research Cambridge Biomedical Research Centre, Eli Lilly and Company, Alexion Pharmaceuticals, and Addenbrooke's Charitable Trust.

CircEPSTI1 in peripheral blood as a novel potential biomarker for childhood‐onset systemic lupus erythematosus

AbstractBackgroundCircular RNA (circRNA) plays an important role in the pathogenesis of many diseases and can be used as a biomarker for diagnosis or disease monitoring. However, reports on circRNA in childhood‐onset systemic lupus erythematosus (cSLE) are limited. Therefore, this study aimed to investigate circEPSTI1 expression in cSLE and evaluate its potential as a biomarker for diagnosing cSLE.MethodsThis study included 70 children diagnosed with cSLE, 20 diagnosed with juvenile idiopathic arthritis (JIA), 20 diagnosed with juvenile dermatomyositis (JDM), and 50 healthy children at the Rheumatology Department of Beijing Children's Hospital from January 2019 to December 2019. Quantitative polymerase chain reaction was used to determine circEPSTI1 expression in the children. Correlations between circEPSTI1 and clinical features were assessed using Spearman's correlation test. Additionally, we calculated the receiver operating characteristic curve to assess the diagnostic efficacy.ResultsWe found that circEPSTI1 expression was higher in children with cSLE (4.62 ± 3.55) than that in healthy children (1.00 ± 0.45), those with JDM (1.06 ± 0.76), and those with JIA (0.96 ± 0.48). The area of the curve of circEPSTI1 was 0.892 (95% confidence interval [CI]: 0.832–0.952, p < 0.001) to discriminate children with SLE from healthy children, with a specificity of 0.814 and a sensitivity of 0.922. Children with lupus nephritis showed a higher circEPSTI1 expression than healthy children, those with JDM, and those with JIA. In addition, circEPSTI1 expression in children with SLE showed significant correlations with the SLE Disease Activity Index (p < 0.0001) and C3 concentrations (p = 0.001).ConclusionOur study suggests that circEPSTI1 is a promising biomarker for the diagnosis and monitoring of cSLE.

Rhodium(III)-Catalyzed C–H Activation in Indole: A Comprehensive Report (2017–2022)

AbstractIn the realm of synthetic organic chemistry, the catalysis of directed C–H activation by transition metals is an outstanding and efficient method for the synthesis of natural products, organic materials, and fundamental organic building blocks. Notably, this strategy has experienced remarkable advances in recent years, particularly in its application to various substrate classes, including the essential indole scaffold. Indole is a highly sought-after target in organic chemistry. The significance of indole extends beyond its use in total synthesis and drug discovery. It also serves as an important tool in the development of pharmaceutical agents, agrochemicals, and materials. By targeting indole, synthetic chemists can access a wide range of bioactive compounds, which opens new avenues for drug development and chemical biology research. The synthesis of structurally varied indoles has been greatly aided by the development of a comprehensive toolkit made possible by the use of C–H activation as a versatile functionalization platform. This review highlights the latest breakthroughs in rhodium-catalyzed C–H activation at the C2, C4, and C7 positions of the indole scaffold. These developments represent significant progress in the field and hold promising potential for further advances in the synthesis of indole-based compounds.1 Introduction2 The Development of Rhodium-Catalyzed C–H Activation3 General Mechanistic Introduction to Rh(III)-Catalyzed C–H Activation4 Direct C–H Functionalization of Indoles4.1 C2 Activation of Indoles4.2 C4 Activation of Indoles4.3 Dual C–H Activation Strategy4.4 C7 Activation of Indoles5 Conclusion

Influence of Dietary Chitosan Supplementation on Growth Indicators, Nutrient Digestibility, Immunity, Cecal Microbiota, and Intestinal Morphology of Growing Male Rabbits

Abstract The present study aimed to assess the influence of dietary chitosan on production performance, nutrient digestibility, slaughter traits, immune function, cecal microbiota, and intestinal histomorphometry of growing rabbits for 56 days. In total, sixty New Zealand White male rabbits aged 35 days (574±7.26 g, mean ± SEM) were arbitrarily assigned to four dietary groups. The experimental diets were fortified with chitosan powder at 0, 250, 500, and 1000 mg per kg diet, respectively. It was found that final weight, specific growth rate, daily weight gain, and feed: gain enhanced linearly (P<0.05) with increasing chitosan levels in the diet. No significant differences were found for all slaughter traits, including hot carcass weight, carcass yield, and internal organ weights, besides carcass parts, including head, fore, intermediate and hind parts. Hematocrit percentage (linear, P = 0.024), serum lysozyme activity (linear, P = 0.004), immunoglobulin M, and complement C3 concentrations (linearly, P<0.001) increased with increasing chitosan supplementation levels. The organic matter (P = 0.009), dry matter (P = 0.008), and nitrogen-free extract (P = 0.012) digestibilities were enhanced quadratically by dietary supplementation with chitosan. Cecal lactobacilli, E. coli, coliform and Salmonella spp. populations (linear, P<0.05) decreased in response to chitosan supplementation. All the dietary chitosan levels increased villus width, villus length, and crypt depth (linear, P<0.001), whereas muscular layer thickness decreased (linear, P=0.002). In summary, supplementing the diet of growing rabbits with chitosan can improve growth indicators, immunity, nutrient digestibility, and intestinal morphology, as well as reduce pathogenic cecal bacteria.

P-216 - Radiosynthesis and preliminary evaluation of a novel PET probe for neuronal complement C3 activation imagingRadiosynthesis and preliminary evaluation of a novel PET probe for neuronal complement C3 activation imaging

P-216 - Radiosynthesis and preliminary evaluation of a novel PET probe for neuronal complement C3 activation imagingRadiosynthesis and preliminary evaluation of a novel PET probe for neuronal complement C3 activation imaging

Omicron subvariants illustrate reduced respiratory tissue penetration, cell damage and inflammatory responses in human airway epithelia.

To explore whether the reported lower pathogenicity in infected individuals of variant of concern (VoC) Omicron and its current subvariants compared to VoC Delta may be related to fundamental differences in the initial virus-tissue interaction, we assessed their ability to penetrate, replicate and cause damage in a human 3D respiratory model. For this, we used TEER measurements, real-time PCR, LDH, cytokine and complex confocal imaging analyses. We observed that Delta readily penetrated deep into the respiratory epithelium and this was associated with major tissue destruction, high LDH activity, high viral loads and pronounced innate immune activation as observed by intrinsic C3 activation and IL-6 release at infection sites. In contrast, Omicron subvariants BA.5, BQ.1.1 and BF7 remained superficially in the mucosal layer resulting merely in outward-directed destruction of cells, maintenance of epithelial integrity, minimal LDH activity and low basolateral release of virus at infection sites, as well as significantly smaller areas of complement activation and lower IL-6 secretion. Interestingly, also within Omicron subvariants differences were observed with newer Omicron subvariants BQ.1.1 and BF.7 illustrating significantly reduced viral loads, IL-6 release and LDH activity compared to BA.5. Our data indicate that earliest interaction events after SARS-CoV-2 transmission may have a role in shaping disease severity.

Effects of dietary sodium butyrate on growth performance, immune function, and intestinal microflora of Chinese soft-shelled turtle (Pelodiscus sinensis).

The Chinese soft-shelled turtle (Pelodiscus sinensis) has become increasingly susceptible to frequent diseases with the intensification of farming, which severely impacts the development of the aquaculture industry. Sodium butyrate (SB) is widely used as a feed additive due to its promotion of growth, enhancement of immune function, and antioxidative properties. This study aimed to investigate the effects of dietary SB on the growth performance, immune function, and intestinal microflora of Chinese soft-shelled turtles. A total of 300 Chinese soft-shelled turtles (mean weight: 11.36 ± 0.21g) were randomly divided into four groups with three parallel sets in each group. Each group was fed a diet supplemented with 0%, 0.005%, 0.01%, or 0.02% SB for 60 days. The results demonstrated an upward trend in weight gain rate (WGR) and specific growth rate (SGR) with increasing SB supplementation, and the experimental group fed with 0.02% SB showed a significant increase in WGR and SGR compared to other groups (P< 0.05). These levels of SB also decreased the levels of feed conversion ratio (FCR) and the total cholesterol (TC) content of Chinese soft-shelled turtles, and the 0.02% SB was significantly lower than that of other groups (P< 0.05). The activity of complement protein in vivo increased with increases in SB content, and the activities of complement C3 and C4 reached the highest level with 0.02% SB. The species abundance of the experimental group D fed with 0.02% SB was significantly higher than that of other groups (P< 0.05). Furthermore, the relative abundance of Clostridium sensu stricto 1 was significantly increased with 0.02% SB (P< 0.05). In conclusion, adding 0.02% SB to the diet improves the growth performance, feed digestion ability, and intestinal microbiota of Chinese soft-shelled turtles.

The Adr1 transcription factor directs regulation of the ergosterol pathway and azole resistance in Candida albicans.

Research often relies on well-studied orthologs within related species, with researchers using a well-studied gene or protein to allow prediction of the function of the ortholog. In the opportunistic pathogen Candida albicans, orthologs are usually compared with Saccharomyces cerevisiae, and this approach has been very fruitful. Many transcription factors (TFs) do similar jobs in the two species, but many do not, and typically changes in function are driven not by modifications in the structures of the TFs themselves but in the connections between the transcription factors and their regulated genes. This strategy of changing TF function has been termed transcription factor rewiring. In this study, we specifically looked for rewired transcription factors, or Candida-specific TFs, that might play a role in drug resistance. We investigated 30 transcription factors that were potentially rewired or were specific to the Candida clade. We found that the Adr1 transcription factor conferred resistance to drugs like fluconazole, amphotericin B, and terbinafine when activated. Adr1 is known for fatty acid and glycerol utilization in Saccharomyces, but our study reveals that it has been rewired and is connected to ergosterol biosynthesis in Candida albicans.

Shade effect on the productivity and structure of natural campos communities: Doctoral thesis abstract

The characteristics of the forest plantations determine the dynamics of photosynthetically active radiation (PAR) under the tree canopy and its area of influence. The objective of this work was to study the effect of shade on the structure and biomass production of the natural grassland (CN), analyzing the relationships between the light environment on the vegetation cover of the understory (%), the species present and their photosynthetic metabolisms. In addition, the effect of artificial shade on the production, seasonal distribution of forage and structure of a CN were analyzed for three years. P. taeda and E. grandis exhibited a similar light environment, presenting the lowest levels of plant cover. E. globulus showed a more illuminated environment, generating better conditions for plant development. C4 species increase their coverage against increases in incident photosynthetically active radiation (PARi) levels and C3 species achieve the highest coverage at intermediate levels of light interception. The composition in summer and autumn tends to become homogeneous over the years, associating with summer perennial grasses (GPE), regardless of the level of interception of solar radiation (IRS). In winter and spring the cover evolves towards a greater association with winter perennial grasses (GPI) and winter annual grasses (GAI) in the levels of greater shade. The increase in IRS levels increased the production of CN. The effect of shade over time determined different seasonal responses in biomass production and composition of functional types of plants. The shade modifies the botanical composition, increasing the coverage of GPI, GAI and reducing the GPE. The shade level modifies the seasonal distribution of forage, smoothing the annual production curve.

Viral-like TLR3 induction of cytokine networks and α-synuclein are reduced by complement C3 blockade in mouse brain

Inflammatory processes and mechanisms are of central importance in neurodegenerative diseases. In the brain, α-synucleinopathies such as Parkinson’s disease (PD) and Lewy body dementia (LBD) show immune cytokine network activation and increased toll like receptor 3 (TLR3) levels for viral double-stranded RNA (dsRNA). Brain inflammatory reactions caused by TLR3 activation are also relevant to understand pathogenic cascades by viral SARS-CoV-2 infection causing post- COVID-19 brain-related syndromes. In the current study, following regional brain TLR3 activation induced by dsRNA in mice, an acute complement C3 response was seen at 2 days. A C3 splice-switching antisense oligonucleotide (ASO) that promotes the splicing of a non-productive C3 mRNA, prevented downstream cytokines, such as IL-6, and α-synuclein changes. This report is the first demonstration that α-synuclein increases occur downstream of complement C3 activation. Relevant to brain dysfunction, post-COVID-19 syndromes and pathological changes leading to PD and LBD, viral dsRNA TLR3 activation in the presence of C3 complement blockade further revealed significant interactions between complement systems, inflammatory cytokine networks and α-synuclein changes.

143 A targeted binding and activation of native C3 without proteolytic cleavage induced by contact with biosurfaces

143 A targeted binding and activation of native C3 without proteolytic cleavage induced by contact with biosurfaces

212 Determinants resulting in high residual C5 activity in presence of Eculizumab or Coversin

212 Determinants resulting in high residual C5 activity in presence of Eculizumab or Coversin

Deletion of complement factor 5 amplifies glucose intolerance in obese male but not female mice.

Complement factor 5 of the innate immune system generates C5a and C5b ligands, which initiate inflammatory and cell lysis events, respectively. C5 activation has been linked with obesity-associated metabolic disorders; however, whether it has a causative role is unclear. We generated a C5 null (C5-/-) mouse using CRISPR-Cas9 gene editing to determine whether loss of C5 improves obesity-linked metabolic dysfunction. Generation of a new mouse model was prompted in part by the observation of off-target gene mutations in commercially available C5-/- lines. Male and female wild-type (WT), heterozygous (Het), and C5-/- mice were fed low-fat diet (LFD) or high-fat diet (HFD) for 22 wk. Body weight gain did not differ between genotypes on LFD or HFD. In lean animals, male C5-/- mice had similar glucose tolerance compared with WT controls; however, in obese conditions, glucose tolerance was worsened in C5-/- compared with controls. In contrast, female mice did not exhibit differences in glucose tolerance between genotypes under either dietary paradigm. Fasting insulin was not different between genotypes, whereas diet-induced obese male C5-/- mice had lower fed insulin concentrations compared with WT controls. No differences in adipose tissue inflammation or adipocyte size were identified between groups. Similarly, susceptibility to fatty liver and hepatic inflammation was similar between WT and C5-/- mice. However, the systemic cytokine response to acute endotoxin exposure was decreased in C5-/- mice. Together, these data suggest that loss of C5 worsens glucose tolerance in obese male but not female mice. Additional work is required to pinpoint the mechanisms by which loss of C5 amplifies glucose intolerance in obesity.NEW & NOTEWORTHY We generated a new mouse model of complement factor 5 deficiency. This work was prompted by a need for improved transgenic mouse lines of C5, due to off-target gene mutations. We find that loss of C5 worsens glucose tolerance in a sex-dependent manner. Though the mechanisms evoking glucose intolerance are not clear, we are confident this model will be useful in interrogating complement activation in obesity-associated diseases.

Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis

Complement factor H (CFH) negatively regulates consumption of complement component 3 (C3), thereby restricting complement activation. Genetic variants in CFH predispose to chronic inflammatory disease. Here, we examined the impact of CFH on atherosclerosis development. In a mouse model of atherosclerosis, CFH deficiency limited plaque necrosis in a C3-dependent manner. Deletion of CFH in monocyte-derived inflammatory macrophages propagated uncontrolled cell-autonomous C3 consumption without downstream C5 activation and heightened efferocytotic capacity. Among leukocytes, Cfh expression was restricted to monocytes and macrophages, increased during inflammation, and coincided with the accumulation of intracellular C3. Macrophage-derived CFH was sufficient to dampen resolution of inflammation, and hematopoietic deletion of CFH in atherosclerosis-prone mice promoted lesional efferocytosis and reduced plaque size. Furthermore, we identified monocyte-derived inflammatory macrophages expressing C3 and CFH in human atherosclerotic plaques. Our findings reveal a regulatory axis wherein CFH controls intracellular C3 levels of macrophages in a cell-autonomous manner, evidencing the importance of on-site complement regulation in the pathogenesis of inflammatory diseases.

Synthesis of Gentamicins C1, C2, and C2a and Antiribosomal and Antibacterial Activity of Gentamicins B1, C1, C1a, C2, C2a, C2b, and X2.

Complementing our earlier syntheses of the gentamicins B1, C1a, C2b, and X2, we describe the synthesis of gentamicins C1, C2, and C2a characterized by methyl substitution at the 6'-position, and so present an alternative access to previous chromatographic methods for accessing these sought-after compounds. We describe the antiribosomal activity of our full set of synthetic gentamicin congeners against bacterial ribosomes and hybrid ribosomes carrying the decoding A site of the human mitochondrial, A1555G mutant mitochondrial, and cytoplasmic ribosomes and establish structure-activity relationships with the substitution pattern around ring I to antiribosomal activity, antibacterial resistance due to the presence of aminoglycoside acetyl transferases acting on the 6'-position in ring I, and literature cochlear toxicity data.