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Microglia, the immune cells of the central nervous system, are dynamic and heterogenous cells. While single cell RNA sequencing has become the conventional methodology for evaluating microglial state, transcriptomics do not provide insight into functional changes, identifying a critical gap in the field. Here, we propose a novel organelle phenotyping approach in which we treat live human induced pluripotent stem cell-derived microglia (iMGL) with organelle dyes staining mitochondria, lipids, lysosomes and acquire data by live-cell spectral microscopy. Dimensionality reduction techniques and unbiased cluster identification allow for recognition of microglial subpopulations with single-cell resolution based on organelle function. We validated this methodology using lipopolysaccharide and IL-10 treatment to polarize iMGL to an "inflammatory" and "anti-inflammatory" state, respectively, and then applied it to identify a novel regulator of iMGL function, complement protein C1q. While C1q is traditionally known as the initiator of the complement cascade, here we use organelle phenotyping to identify a role for C1q in regulating iMGL polarization via fatty acid storage and mitochondria membrane potential. Follow up evaluation of microglia using traditional read outs of activation state confirm that C1q drives an increase in microglia pro-inflammatory gene production and migration, while suppressing microglial proliferation. These data together validate the use of a novel organelle phenotyping approach and enable better mechanistic investigation of molecular regulators of microglial state.

IntroductionSynaptic loss is a hallmark of Alzheimer's disease (AD) that correlates with cognitive decline in AD patients. Complement-mediated synaptic pruning has been associated with this excessive loss of synapses in AD. Here, we investigated the effect of C5aR1 inhibition on microglial and astroglial synaptic pruning in two mouse models of AD.MethodsA combination of super-resolution and confocal and tridimensional image reconstruction was used to assess the effect of genetic ablation or pharmacological inhibition of C5aR1 on the Arctic48 and Tg2576 models of AD.ResultsGenetic ablation or pharmacological inhibition of C5aR1 partially rescues excessive pre-synaptic pruning and synaptic loss in an age and region-dependent fashion in two mouse models of AD, which correlates with improved long-term potentiation (LTP).DiscussionReduction of excessive synaptic pruning is an additional beneficial outcome of the suppression of C5a-C5aR1 signaling, further supporting its potential as an effective targeted therapy to treat AD.HighlightsC5aR1 ablation restores long-term potentiation in the Arctic model of AD. C5aR1 ablation rescues region specific excessive pre-synaptic loss. C5aR1 antagonist, PMX205, rescues VGlut1 loss in the Tg2576 model of AD. C1q tagging is not sufficient to induce VGlut1 microglial ingestion. Astrocytes contribute to excessive pre-synaptic loss at late stages of the disease.

Background: The CD2-associated protein (CD2AP) was initially identified in peripheral immune cells and regulates cytoskeleton and protein trafficking. Single nucleotide polymorphisms (SNPs) in the CD2AP gene have been associated with Alzheimer's disease (AD). However, the functional role of CD2AP, especially its role in microglia during AD onset, remains elusive. Methods: CD2AP protein levels in cultured primary cells and in 5xFAD mice was studied. Microglial CD2AP-deficient mice were crossed with 5xFAD mice and the offspring were subjected to neuropathological assessment, behavioral tests, electrophysiology, RNA-seq, Golgi staining, and biochemistry analysis. Primary microglia were also isolated for assessing their uptake and morphology changes. Results: We find that CD2AP is abundantly expressed in microglia and its levels are elevated in the brain of AD patients and the 5xFAD model mice at pathological stages. We demonstrate that CD2AP haploinsufficiency in microglia significantly attenuates cognitive and synaptic deficits, weakens the response of microglia to Aβ and the formation of disease-associated microglia (DAM), and alleviates synapse loss in 5xFAD mice. We show that CD2AP-deficient microglia exhibit compromised uptake ability. In addition, we find that CD2AP expression is positively correlated with the expression of the complement C1q that is important for synapse phagocytosis and the formation of DAM in response to Aβ deposition. Moreover, we reveal that CD2AP interacts with colony stimulating factor 1 receptor (CSF1R) and regulates CSF1R cell surface levels, which may further affect C1q expression. Conclusions: Our results demonstrate that CD2AP regulates microgliosis and identify a protective function of microglial CD2AP deficiency against Aβ deposition, suggesting the importance of detailed investigation of AD-associated genes in different brain cells for thoroughly understanding their exact contribution to AD. [ABSTRACT FROM AUTHOR]

Human C1q is a multifaceted complement protein whose functions range from activating the complement classical pathway to immunomodulation and promoting placental development and tumorigenesis. It is encoded by the C1QA , C1QB , and C1QC genes located on chromosome 1. C1q, unlike most complement components, has extrahepatic expression by a range of cells including macrophages, monocytes and immature dendritic cells. Its local synthesis under the conditions of inflammation and for the purpose of removal of altered self requires its strict transcriptional regulation. To delve into C1Q transcriptional regulation and unravel potential epigenetic influences, we conducted an in silico analysis utilizing a range of online tools and datasets. Co-expression analysis revealed tight coordination between C1QA , C1QB , and C1QC genes. Strikingly, distinct epigenetic patterns emerged across various cell types expressing or lacking these genes, with unique histone marks and DNA methylation status characterizing their regulatory landscape. Notably, the investigation extended to tumor contexts, unveiled potential epigenetic roles in malignancies. The cell type and tumor-specific histone modifications and chromatin accessibility patterns underscore the dynamic nature of epigenetic regulation of C1Q , providing crucial insights into the intricate mechanisms governing the expression of these immunologically significant genes. The findings provide a foundation for future investigations into targeted epigenetic modulation, offering insights into potential therapeutic avenues for immune-related disorders and cancer mediated via C1q. [ABSTRACT FROM AUTHOR]

Background: Sepsis‐associated encephalopathy (SAE) is a serious neurologic complication in septic patients with poor prognoses. There is increasing evidence that stimulator of interferon genes (STING) plays a crucial role in neuroinflammation and cognitive impairment. However, whether sepsis associated with STING changes contributes to cognitive impairment is unknown. Methods: Male adult mice received lipopolysaccharide (LPS) injection (a single dose of 4 mg/kg; i.p. injection) and 30 min later, they were injected with STING inhibitor C‐176 (a single dose of 30 mg/kg, i.p. injection). Behavioral assessments, biochemical measurements, in vivo and ex vivo electrophysiology techniques were conducted to investigate the association between LPS‐induced STING overexpression and cognitive function. Results: Cognitive impairment was associated with STING overexpression and activation of microglia/macrophages. Phagocytosis of microglia/macrophages as well as complement C1q release were increased after LPS injection, leading to abnormal pruning synapses, synaptic transmission reduction, long‐term potentiation (LTP) impairment, as well as abnormal theta oscillation in the hippocampus. Notably, STING inhibitor C‐176 significantly reversed these changes. Conclusions: Sepsis‐induced STING overexpression in microglia/macrophages may lead to synaptic loss, abnormal theta oscillation and LTP impairment through microglia/macrophages activation and complement C1q modulation, ultimately resulting in cognitive impairment. [ABSTRACT FROM AUTHOR]

Feline infectious peritonitis (FIP) is a fatal disease in cats caused by infection with feline coronavirus (FCoV). Despite severe inflammatory changes, defense mechanisms fail to achieve virus clearance. Some studies focused on various immune evasion mechanisms, but none of these studies elucidated the inefficacy of the complement system, which is one major player in FIP-associated immune pathogenesis. This study aimed to investigate the involvement of complement-regulating factors (CRFs). CRFs help modulate the immune response and prevent host tissue damage. Archived tissue samples from 31 deceased, FIP-affected cats were evaluated using multiplex immunohistochemistry for the spatial expression of the complement-regulating factors CD46 and CD59 in association with FIP lesions and their colocalization with complement-activating factor C1q and membrane attack complex C9 in relation to the presence and proximity of FCoV-infected cells. The FIP lesions of all 31 cats exhibited marked expression of both complement-regulating factors in proximity to FCoV-infected macrophages. Moreover, their expression in all 31 animals was significantly lower than the expression of the complement-activating factors C1q and C9 compared to areas farther distal to FCoV-infected cells. In conclusion, FCoV-infected macrophages in cats with FIP appear to use autocrine and paracrine expression of complement-regulating factors in their immediate environment to shield themselves from destruction by the complement system. [ABSTRACT FROM AUTHOR]

Normal aging, though lacking widespread neurodegeneration, is nevertheless characterized by cognitive impairment in learning, memory, and executive function. The aged brain is spared from neuron loss, but white matter is lost and damage to myelin sheaths accumulates. This myelin damage is strongly associated with cognitive impairment. Although the cause of the myelin damage is not known, microglia dysregulation is a likely contributor. Immunologic proteins interact with microglial receptors to modulate microglia-mediated phagocytosis, which mediates myelin damage clearance and turn-over. Two such proteins, "eat me" signal C1q and "don't eat me" signal CD47, act in opposition with microglia. Both C1q and CD47 have been implicated in Multiple Sclerosis, a demyelinating disease, but whether they play a role in age-related myelin pathology is currently unknown. The present study investigates C1q and CD47 in relation to age-related myelin degeneration using multilabel immunofluorescence, RNAscope, and confocal microscopy in the cingulum bundle of male and female rhesus monkeys across the lifespan. Our findings showed significant age-related elevation in C1q localized to myelin basic protein, and this increase is associated with more severe cognitive impairment. In contrast, CD47 localization to myelin decreased in middle age and oligodendrocyte expression of CD47 RNA decreased with age. Lastly, microglia reactivity increased with age in association with the changes in C1q and CD47. Together, these results suggest disruption in the balance of "eat me" and "don't eat me" signals during normal aging, biasing microglia toward increased reactivity and phagocytosis of myelin, resulting in cognitive deficits. [ABSTRACT FROM AUTHOR]

Abstract Background The CD2-associated protein (CD2AP) was initially identified in peripheral immune cells and regulates cytoskeleton and protein trafficking. Single nucleotide polymorphisms (SNPs) in the CD2AP gene have been associated with Alzheimer’s disease (AD). However, the functional role of CD2AP, especially its role in microglia during AD onset, remains elusive. Methods CD2AP protein levels in cultured primary cells and in 5xFAD mice was studied. Microglial CD2AP-deficient mice were crossed with 5xFAD mice and the offspring were subjected to neuropathological assessment, behavioral tests, electrophysiology, RNA-seq, Golgi staining, and biochemistry analysis. Primary microglia were also isolated for assessing their uptake and morphology changes. Results We find that CD2AP is abundantly expressed in microglia and its levels are elevated in the brain of AD patients and the 5xFAD model mice at pathological stages. We demonstrate that CD2AP haploinsufficiency in microglia significantly attenuates cognitive and synaptic deficits, weakens the response of microglia to Aβ and the formation of disease-associated microglia (DAM), and alleviates synapse loss in 5xFAD mice. We show that CD2AP-deficient microglia exhibit compromised uptake ability. In addition, we find that CD2AP expression is positively correlated with the expression of the complement C1q that is important for synapse phagocytosis and the formation of DAM in response to Aβ deposition. Moreover, we reveal that CD2AP interacts with colony stimulating factor 1 receptor (CSF1R) and regulates CSF1R cell surface levels, which may further affect C1q expression. Conclusions Our results demonstrate that CD2AP regulates microgliosis and identify a protective function of microglial CD2AP deficiency against Aβ deposition, suggesting the importance of detailed investigation of AD-associated genes in different brain cells for thoroughly understanding their exact contribution to AD.

Background: Tuberculosis (TB) poses a significant public health challenge, particularly because it can exist in an asymptomatic latent phase. Latent TB infection indicates the presence of Mycobacterium tuberculosis without clinical symptoms. Effectively distinguishing between active and latent TB is essential, especially in regions with high TB prevalence, as it may help reduce transmission rates. This study aims to evaluate C1q as a potential biomarker for differentiating active TB from latent forms. Methods: This prospective cross-sectional study was conducted from January 2017 to February 2018, involving HIV-negative adults aged 18 and older attending TB clinics. Participants were categorized based on clinical symptoms, imaging results, and laboratory tests into active or latent TB. Blood samples were collected to assess serum C1q levels, which were then compared between the two groups. Results: Out of 81 patients referred for TB evaluation, 38 were diagnosed with active TB. The overall median C1q level was 6.46 μg/ml (interquartile range 4.66–10). The active TB group exhibited significantly elevated C1q levels (10.21 μg/ml) compared to the latent TB group (6.03 μg/ml, P < 0.001). The area under the receiver operating characteristic curve for C1q in distinguishing active from latent TB was 0.74 (95% confidence interval, 0.63–0.85), with sensitivity varying between 61% and 82% at different threshold values. Conclusions: C1q shows potential as a reliable and easily obtainable biomarker for differentiating active TB from latent infection, demonstrating high sensitivity. These results underscore the need for further research to explore the clinical application of C1q in TB diagnostics.

Abstract Introduction The role of complement system in late stage of IgA nephropathy (IgAN) remains unknown. We therefore investigated the effects of complement system on worsening kidney function in advanced (stage 4 CKD) IgAN. Methods Renal specimens of 69 IgAN patients who underwent renal biopsy during stage 4 CKD between 2010 and 2021, were stained using immunofluorescence (IF) and immunohistochemistry (IHC) for glomerular complement components. The primary outcome was progression to end-stage renal disease (ESRD). Associations of complement components with baseline clinicopathological characteristics and outcomes were assessed using multivariable Cox regression and Spearman analyses. Results During a median follow-up of 18.0 months, 26 (37.7%) patients progressed to ESRD and none died. C1q and C3 deposition were detected in 12 and 66 patients, respectively. Higher eGFR [hazards ratio (HR), 0.852, 95% confidence interval (CI), 0.756–0.959; P = 0.008], higher C3 intensity (HR, 2.955, 95%CI, 1.063–8.220; P = 0.038) and T1-2 score (HR, 2.576, 95%CI, 1.205–5.576, P = 0.015) were predictive of time to ESRD in CKD 4 stage IgAN. Significant expressions of C1q (P = 0.005), C4d (P

Introduction: The role of complement system in late stage of IgA nephropathy (IgAN) remains unknown. We therefore investigated the effects of complement system on worsening kidney function in advanced (stage 4 CKD) IgAN. Methods: Renal specimens of 69 IgAN patients who underwent renal biopsy during stage 4 CKD between 2010 and 2021, were stained using immunofluorescence (IF) and immunohistochemistry (IHC) for glomerular complement components. The primary outcome was progression to end-stage renal disease (ESRD). Associations of complement components with baseline clinicopathological characteristics and outcomes were assessed using multivariable Cox regression and Spearman analyses. Results: During a median follow-up of 18.0 months, 26 (37.7%) patients progressed to ESRD and none died. C1q and C3 deposition were detected in 12 and 66 patients, respectively. Higher eGFR [hazards ratio (HR), 0.852, 95% confidence interval (CI), 0.756–0.959; P = 0.008], higher C3 intensity (HR, 2.955, 95%CI, 1.063–8.220; P = 0.038) and T1-2 score (HR, 2.576, 95%CI, 1.205–5.576, P = 0.015) were predictive of time to ESRD in CKD 4 stage IgAN. Significant expressions of C1q (P = 0.005), C4d (P < 0.001), factor B (P < 0.001), C3 (P = 0.042) and C5b-9 (P = 0.004) were identified in ESRD group than in non-ESRD group by IHC, while MBL expression was low. Although they were not associated with baseline 24 h-UP, higher factor B and C1q expressions were both correlated with a lower baseline eGFR (P < 0.001 and = 0.04, respectively) and the deterioration of kidney function during follow-up (P = 0.046 and 0.015, respectively). Conclusion: Complement deposition in IgAN patients with stage 4 CKD portends a faster deterioration of kidney function. Activation of classical and alternative complement pathways plays a major role in this stage. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis (TB) poses a significant public health challenge, particularly because it can exist in an asymptomatic latent phase. Latent TB infection indicates the presence of Mycobacterium tuberculosis without clinical symptoms. Effectively distinguishing between active and latent TB is essential, especially in regions with high TB prevalence, as it may help reduce transmission rates. This study aims to evaluate C1q as a potential biomarker for differentiating active TB from latent forms. Methods: This prospective cross-sectional study was conducted from January 2017 to February 2018, involving HIV-negative adults aged 18 and older attending TB clinics. Participants were categorized based on clinical symptoms, imaging results, and laboratory tests into active or latent TB. Blood samples were collected to assess serum C1q levels, which were then compared between the two groups. Results: Out of 81 patients referred for TB evaluation, 38 were diagnosed with active TB. The overall median C1q level was 6.46 μg/ml (interquartile range 4.66–10). The active TB group exhibited significantly elevated C1q levels (10.21 μg/ml) compared to the latent TB group (6.03 μg/ml, P < 0.001). The area under the receiver operating characteristic curve for C1q in distinguishing active from latent TB was 0.74 (95% confidence interval, 0.63–0.85), with sensitivity varying between 61% and 82% at different threshold values. Conclusions: C1q shows potential as a reliable and easily obtainable biomarker for differentiating active TB from latent infection, demonstrating high sensitivity. These results underscore the need for further research to explore the clinical application of C1q in TB diagnostics. [ABSTRACT FROM AUTHOR]

Microglia, the immune cells of the central nervous system, are dynamic and heterogenous cells. While single cell RNA sequencing has become the conventional methodology for evaluating microglial state, transcriptomics do not provide insight into functional changes, identifying a critical gap in the field. Here, we propose a novel organelle phenotyping approach in which we treat live human induced pluripotent stem cell‐derived microglia (iMGL) with organelle dyes staining mitochondria, lipids, lysosomes and acquire data by live‐cell spectral microscopy. Dimensionality reduction techniques and unbiased cluster identification allow for recognition of microglial subpopulations with single‐cell resolution based on organelle function. We validated this methodology using lipopolysaccharide and IL‐10 treatment to polarize iMGL to an "inflammatory" and "anti‐inflammatory" state, respectively, and then applied it to identify a novel regulator of iMGL function, complement protein C1q. While C1q is traditionally known as the initiator of the complement cascade, here we use organelle phenotyping to identify a role for C1q in regulating iMGL polarization via fatty acid storage and mitochondria membrane potential. Follow up evaluation of microglia using traditional read outs of activation state confirm that C1q drives an increase in microglia pro‐inflammatory gene production and migration, while suppressing microglial proliferation. These data together validate the use of a novel organelle phenotyping approach and enable better mechanistic investigation of molecular regulators of microglial state. [ABSTRACT FROM AUTHOR]

Stroke is one of the major causes of disability and death worldwide. The lack of effective medical treatment for stroke heightens the need for new therapeutic targets. In this study, we obtained two microarray data sets from the Gene Expression Omnibus (GEO) database and identified differential genes (DEGs) between MCAO and control groups. Then, enrichment analysis of the DEGs was performed using DAVID and Metascape. The results show 27 DEGs shared between the two datasets. The functional enrichment analysis showed that these genes are mainly enriched in immune response, complement and coagulation cascades, apoptotic processes. The four hub genes (C1qc, Fcgr2b, C1qb, and Cd14) were screened out using the Cytoscape. Next, real-time PCR and Western blot analysis showed that expression of C1q and CD14 increased at 14 days after tMCAO. Furthermore, we took eight small molecule compounds with the lowest score using Cmap and studied their background characteristics. These results are built on a meta-analysis of data, which are generally accessible from the online space. Finally, we evaluated the protective effect of the rolipram through behavior tests after tMCAO, and results showed that the rolipram significantly attenuated neurobehavioral dysfunction at 14 days after brain ischemia. The present results provide novel insights into the biological process and potential therapeutic drugs involved in stroke. [ABSTRACT FROM AUTHOR]

Immunoglobulin G (IgG) molecules that bind antigens on the membrane of target cells spontaneously form hexameric rings, thus recruiting C1 to initiate the complement pathway. However, our previous report indicated that a mouse IgG mutant lacking the Cγ1 domain activates the pathway independently of antigen presence through its monomeric interaction with C1q via the CL domain, as well as Fc. In this study, we investigated the potential interaction between C1q and human CL isoforms. Quantitative single-molecule observations using high-speed atomic force microscopy revealed that human Cκ exhibited comparable C1q binding capabilities with its mouse counterpart, surpassing the Cλ types, which have a higher isoelectric point than the Cκ domains. Nuclear magnetic resonance and mutation experiments indicated that the human and mouse Cκ domains share a common primary binding site for C1q, centred on Glu194, a residue conserved in the Cκ domains but absent in the Cλ domains. Additionally, the Cγ1 domain, with its high isoelectric point, can cause electrostatic repulsion to the C1q head and impede the C1q-interaction adjustability of the Cκ domain in Fab. The removal of the Cγ1 domain is considered to eliminate these factors and thus promote Cκ interaction with C1q with the potential risk of uncontrolled activation of the complement pathway in vivo in the absence of antigen. However, this research underscores the presence of potential subsites in Fab for C1q binding, offering promising targets for antibody engineering to refine therapeutic antibody design. [ABSTRACT FROM AUTHOR]

Human C1q is a multifaceted complement protein whose functions range from activating the complement classical pathway to immunomodulation and promoting placental development and tumorigenesis. It is encoded by the C1QA, C1QB, and C1QC genes located on chromosome 1. C1q, unlike most complement components, has extrahepatic expression by a range of cells including macrophages, monocytes and immature dendritic cells. Its local synthesis under the conditions of inflammation and for the purpose of removal of altered self requires its strict transcriptional regulation. To delve into C1Q transcriptional regulation and unravel potential epigenetic influences, we conducted an in silico analysis utilizing a range of online tools and datasets. Co-expression analysis revealed tight coordination between C1QA, C1QB, and C1QC genes. Strikingly, distinct epigenetic patterns emerged across various cell types expressing or lacking these genes, with unique histone marks and DNA methylation status characterizing their regulatory landscape. Notably, the investigation extended to tumor contexts, unveiled potential epigenetic roles in malignancies. The cell type and tumor-specific histone modifications and chromatin accessibility patterns underscore the dynamic nature of epigenetic regulation of C1Q, providing crucial insights into the intricate mechanisms governing the expression of these immunologically significant genes. The findings provide a foundation for future investigations into targeted epigenetic modulation, offering insights into potential therapeutic avenues for immune-related disorders and cancer mediated via C1q.

Abstract Background In the past few years, circulating complement C1q involvement in atherosclerosis has garnered growing research interest in addition to the emerging recognition of the novel lipid marker named atherogenic index of plasma (AIP). Nevertheless, among patients experiencing low-density lipoprotein cholesterol (LDL-C) levels less than 1.8mmol/L, the interplay between C1q combined with the AIP for coronary artery disease (CAD) is ambiguous. Methods Patients were stratified into a non-CAD and CAD group according to their coronary angiography. The association between C1q in conjunction with the AIP and CAD was explored using restricted cubic spline analyses and logistic regression models. To assess how it predicted, a receiver operating characteristic analysis was undertaken. Results A total of 7270 patients comprised 1476 non-CAD patients and 5794 patients diagnosed with CAD were analyzed. A comparison of the two groups showed that the C1q levels were notably higher compared to the CAD group, while AIP exhibited an inverse trend. Across quartiles of C1q, the AIP demonstrated a decline with increasing C1q levels, and significant differences were observed between the groups. A correlation analysis underscored a notable negative correlation between the two variables. Univariate and multivariate logistic regression analyses revealed significant associations between CAD and the C1q quartile groups/AIP. Furthermore, compared with the Q4 group, a decrease in the C1q levels corresponded to an escalation in CAD risk, with the odds ratio rising from 1.661 to 2.314. Conclusions In conclusion, there appears to be a notable positive correlation between the combination of C1q with the AIP and CAD.

Endometriosis (EM) is defined as the engraftment and proliferation of functional endometrial-like tissue outside the uterine cavity, leading to a chronic inflammatory condition. While the precise etiology of EM remains elusive, recent studies have highlighted the crucial involvement of a dysregulated immune system. The complement system is one of the predominantly altered immune pathways in EM. Owing to its involvement in the process of angiogenesis, here, we have examined the possible role of the first recognition molecule of the complement classical pathway, C1q. C1q plays seminal roles in several physiological and pathological processes independent of complement activation, including tumor growth, placentation, wound healing, and angiogenesis. Gene expression analysis using the publicly available data revealed that C1q is expressed at higher levels in EM lesions compared to their healthy counterparts. Immunohistochemical analysis confirmed the presence of C1q protein, being localized around the blood vessels in the EM lesions. CD68+ macrophages are the likely producer of C1q in the EM lesions since cultured EM cells did not produce C1q in vitro. To explore the underlying reasons for increased C1q expression in EM, we focused on its established pro-angiogenic role. Employing various angiogenesis assays on primary endothelial endometriotic cells, such as migration, proliferation, and tube formation assays, we observed a robust proangiogenic effect induced by C1q on endothelial cells in the context of EM. C1q promoted angiogenesis in endothelial cells isolated from EM lesions (as well as healthy ovary that is also rich in C1q). Interestingly, endothelial cells from EM lesions seem to overexpress the receptor for the globular heads of C1q (gC1qR), a putative C1q receptor. Experiments with siRNA to silence gC1qR resulted in diminished capacity of C1q to perform its angiogenic functions, suggesting that C1q is likely to engage gC1qR in the pathophysiology of EM. gC1qR can be a potential therapeutic target in EM patients that will disrupt C1q-mediated proangiogenic activities in EM. [ABSTRACT FROM AUTHOR]

Nephritis is a frequent and severe complication of Systemic Lupus Erythematous (SLE). The clinical course of lupus nephritis (LN) is usually characterized by alternating phases of remission and exacerbation. Flares of LN can lead to deterioration of kidney function, necessitating timely diagnosis and therapy. The presence of autoantibodies against C1q (anti-C1qAb) in the sera of SLE patients has been reported in various studies. Some research suggests that the presence and changes in the titer of anti-C1qAb may be associated with the development of LN, as well as with LN activity and renal flares. However, the exact role of anti-C1qAb in LN remains a subject of debate. Despite variability in the results of published studies, anti-C1qAb hold promise as noninvasive markers for assessing LN activity in SLE patients. Measuring anti-C1qAb levels could aid in diagnosing and managing LN during periods of both inactive disease and renal flares. Nevertheless, larger controlled trials with standardized laboratory assays are necessary to further establish the utility of anti-C1qAb in predicting the reactivation and remission of LN and guiding treatment strategies. [ABSTRACT FROM AUTHOR]

The classical pathway of the complement system is activated by the binding of C1q in the C1 complex to the target activator, including immune complexes. Factor H is regarded as the key downregulatory protein of the complement alternative pathway. However, both C1q and factor H bind to target surfaces via charge distribution patterns. For a few targets, C1q and factor H compete for binding to common or overlapping sites. Factor H, therefore, can effectively regulate the classical pathway activation through such targets, in addition to its previously characterized role in the alternative pathway. Both C1q and factor H are known to recognize foreign or altered-self materials, e.g., bacteria, viruses, and apoptotic/necrotic cells. Clots, formed by the coagulation system, are an example of altered self. Factor H is present abundantly in platelets and is a well-known substrate for FXIIIa. Here, we investigated whether clots activate the complement classical pathway and whether this is regulated by factor H. We show here that both C1q and factor H bind to the fibrin formed in microtiter plates and the fibrin clots formed under in vitro physiological conditions. Both C1q and factor H become covalently bound to fibrin clots, and this is mediated via FXIIIa. We also show that fibrin clots activate the classical pathway of complement, as demonstrated by C4 consumption and membrane attack complex detection assays. Thus, factor H downregulates the activation of the classical pathway induced by fibrin clots. These results elucidate the intricate molecular mechanisms through which the complement and coagulation pathways intersect and have regulatory consequences. [ABSTRACT FROM AUTHOR]

PurposeMethodsResultsConclusionsTo assess the clinical relevance of pathophysiology-based biomarkers, specifically serum C1q and whole blood interferon gene signature score (IGSS), in ocular tuberculosis (OTB) diagnosis by conducting an integrative analysis of clinical presentations and treatment response.This retrospective cohort study analysed data from 70 patients with suspected OTB at a tertiary care uveitis practice in Indonesia. Serum C1q levels and whole blood IGSS were quantified. Patients were categorized into four quadrants based on their biomarker profiles: quadrant 1 (high C1q & low IGSS), quadrant 2 (high C1q & high IGSS), quadrant 3 (low C1q & high IGSS), and quadrant 4 (low C1q & low IGSS). Characteristics of clinical presentations, work-up results, and treatment outcomes were explored according to the predefined quadrants.We identified that the majority of OTB patients diagnosed with concurrent active pulmonary TB were in quadrant 1, 2, or 3 (20/23, 87.0%). Twenty-seven patients (27/47, 57.4%) with clinically undifferentiated uveitis were in quadrant 4 (p < 0.001). Among patients in quadrants 1, 2, and 3, completion of a full course of antitubercular treatment (ATT) was associated with a lower number of patients showing persistence or recurrence of ocular inflammation compared to those who were not fully treated with ATT (14.3% vs 85.7%, p = 0.001).Based on the analysis of clinical features and treatment outcomes, patients with elevated levels of either or both serum C1q and whole blood IGSS may reflect active TB disease in the eye, necessitating full ATT management. [ABSTRACT FROM AUTHOR]

Background and Purpose: To deepen our knowledge of the role of complement in synaptic impairment in experimental autoimmune encephalomyelitis (EAE) mice, we investigated the distribution of C1q and C3 proteins and the role of complement as a promoter of glutamate release in purified nerve endings (synaptosomes) and astrocytic processes (gliosomes) isolated from the cortex of EAE mice at the acute stage of the disease (21 ± 1 day post‐immunization). Experimental Approach: EAE cortical synaptosomes and gliosomes were analysed for glutamate release efficiency (measured as release of preloaded [3H]D‐aspartate ([3H]D‐ASP)), C1q and C3 protein density, and for viability and ongoing apoptosis. Key Results: In healthy mice, complement releases [3H]D‐ASP from gliosomes more efficiently than from synaptosomes. The releasing activity occurs in a dilution‐dependent manner and involves the reversal of the excitatory amino acid transporters (EAATs). In EAE mice, the complement‐induced releasing activity is significantly reduced in cortical synaptosomes but amplified in cortical gliosomes. These adaptations are paralleled by decreased density of the EAAT2 protein in synaptosomes and increased EAAT1 staining in gliosomes. Concomitantly, PSD95, GFAP, and CD11b, but not SNAP25, proteins are overexpressed in the cortex of the EAE mice. Similarly, C1q and C3 protein immunostaining is increased in EAE cortical synaptosomes and gliosomes, although signs of ongoing apoptosis or altered viability are not detectable. Conclusion and Implications: Our results unveil a new noncanonical role of complement in the CNS of EAE mice relevant to disease progression and central synaptopathy that suggests new therapeutic targets for the management of MS. [ABSTRACT FROM AUTHOR]

Introduction: Significant neurologic morbidity is caused by pediatric cerebrospinal fluid (CSF) shunt infections. The underlying mechanisms leading to impaired school performance and increased risk of seizures are unknown, however, a better understanding of these mechanisms may allow us to temper their consequences. Recent evidence has demonstrated important roles for complement proteins in neurodevelopment and neuroinflammation. Methods: We examined complement activation throughout Staphylococcus epidermidis (S. epidermidis) central nervous system (CNS) catheter infection. In addition, based on accumulating evidence that C3 plays a role in synaptic pruning in other neuroinflammatory states we determined if C3 and downstream C5 led to alterations in synaptic protein levels. Using our murine model of S. epidermidis catheter infection we quantified levels of the complement components C1q, Factor B, MASP2, C3, and C5 over the course of infection along with bacterial burdens. Results: We found that MASP2 predominated early in catheter infection, but that Factor B was elevated at intermediate time points. Unexpectedly C1q was elevated at late timepoints when bacterial burdens were low or undetectable. Based on these findings and the wealth of information regarding the emerging roles of C1q in the CNS, this suggests functions beyond pathogen elimination during S. epidermidis CNS catheter infection. To identify if C3 impacted synaptic protein levels we performed synaptosome isolation and quantified levels of VGLUT1 and PSD95 as well as pre-, post- and total synaptic puncta in cortical layer V of C3 knockout (KO) andwild typemice. We also used C5 KO andwild typemice to determine if therewas any difference in pre-, post- and total synaptic puncta. Discussion: Neither C3 nor C5 impacted synaptic protein abundance. These findings suggest that chronic elevations in C1q in the brain that persist once CNS catheter infection has resolved may be modulating disease sequalae. [ABSTRACT FROM AUTHOR]

Background: In the past few years, circulating complement C1q involvement in atherosclerosis has garnered growing research interest in addition to the emerging recognition of the novel lipid marker named atherogenic index of plasma (AIP). Nevertheless, among patients experiencing low-density lipoprotein cholesterol (LDL-C) levels less than 1.8mmol/L, the interplay between C1q combined with the AIP for coronary artery disease (CAD) is ambiguous. Methods: Patients were stratified into a non-CAD and CAD group according to their coronary angiography. The association between C1q in conjunction with the AIP and CAD was explored using restricted cubic spline analyses and logistic regression models. To assess how it predicted, a receiver operating characteristic analysis was undertaken. Results: A total of 7270 patients comprised 1476 non-CAD patients and 5794 patients diagnosed with CAD were analyzed. A comparison of the two groups showed that the C1q levels were notably higher compared to the CAD group, while AIP exhibited an inverse trend. Across quartiles of C1q, the AIP demonstrated a decline with increasing C1q levels, and significant differences were observed between the groups. A correlation analysis underscored a notable negative correlation between the two variables. Univariate and multivariate logistic regression analyses revealed significant associations between CAD and the C1q quartile groups/AIP. Furthermore, compared with the Q4 group, a decrease in the C1q levels corresponded to an escalation in CAD risk, with the odds ratio rising from 1.661 to 2.314. Conclusions: In conclusion, there appears to be a notable positive correlation between the combination of C1q with the AIP and CAD. [ABSTRACT FROM AUTHOR]

The interaction between IgM and C1q represents the first step of the classical pathway of the complement system in higher vertebrates. To identify the significance of particular IgM/C1q interactions, recombinant IgMs were used in both hexameric and pentameric configurations and with two different specificities, along with C1q derived from human serum (sC1q) and two recombinant single-chain variants of the trimeric globular region of C1q. Interaction and complement activation assays were performed using the ELISA format, and bio-layer interferometry measurements to study kinetic behavior. The differences between hexameric and pentameric IgM conformations were only slightly visible in the interaction assay, but significant in the complement activation assay. Hexameric IgM requires a lower concentration of sC1q to activate the complement compared to pentameric IgM, leading to an increased release of C4 compared to pentameric IgM. The recombinant C1q mimetics competed with sC1q in interaction assays and were able to inhibit complement activation. The bio-layer interferometry measurements revealed KD values in the nanomolar range for the IgM/C1q interaction, while the C1q mimetics exhibited rapid on and off binding rates with the IgMs. Our results make C1q mimetics valuable tools for developing recombinant C1q, specifically its variants, for further scientific studies and clinical applications. [ABSTRACT FROM AUTHOR]

Normal aging, though lacking widespread neurodegeneration, is nevertheless characterized by cognitive impairment in learning, memory, and executive function. The aged brain is spared from neuron loss, but white matter is lost and damage to myelin sheaths accumulates. This myelin damage is strongly associated with cognitive impairment. Although the cause of the myelin damage is not known, microglia dysregulation is a likely contributor. Immunologic proteins interact with microglial receptors to modulate microglia-mediated phagocytosis, which mediates myelin damage clearance and turn-over. Two such proteins, “eat me” signal C1q and “don’t eat me” signal CD47, act in opposition with microglia. Both C1q and CD47 have been implicated in Multiple Sclerosis, a demyelinating disease, but whether they play a role in age-related myelin pathology is currently unknown. The present study investigates C1q and CD47 in relation to age-related myelin degeneration using multilabel immunofluorescence, RNAscope, and confocal microscopy in the cingulum bundle of male and female rhesus monkeys across the lifespan. Our findings showed significant age-related elevation in C1q localized to myelin basic protein, and this increase is associated with more severe cognitive impairment. In contrast, CD47 localization to myelin decreased in middle age and oligodendrocyte expression of CD47 RNA decreased with age. Lastly, microglia reactivity increased with age in association with the changes in C1q and CD47. Together, these results suggest disruption in the balance of “eat me” and “don’t eat me” signals during normal aging, biasing microglia toward increased reactivity and phagocytosis of myelin, resulting in cognitive deficits.

The contribution of the gut microbiome to neuroinflammation, cognition, and Alzheimer's disease progression has been highlighted over the past few years. Additionally, inhibition of various components of the complement system has repeatedly been demonstrated to reduce neuroinflammation and improve cognitive performance in AD mouse models. Whether the deletion of these complement components is associated with distinct microbiome composition, which could impact neuroinflammation and cognitive performance in mouse models has not yet been examined. Here, we provide a comprehensive analysis of conditional and constitutive knockouts, pharmacological inhibitors, and various housing paradigms for the animal models and wild-type controls at various ages. We aimed to determine the impact of C1q or C5aR1 inhibition on the microbiome in the Arctic and Tg2576 mouse models of AD, which develop amyloid plaques at different ages and locations. Analysis of fecal samples from WT and Arctic mice following global deletion of C1q demonstrated significant alterations to the microbiomes of Arctic but not WT mice, with substantial differences in abundances of Erysipelotrichales, Clostridiales and Alistipes. While no differences in microbiome diversity were detected between cohoused wildtype and Arctic mice with or without the constitutive deletion of the downstream complement receptor, C5aR1, a difference was detected between the C5aR1 sufficient (WT and Arctic) and deficient (C5ar1KO and ArcticC5aR1KO) mice, when the mice were housed segregated by C5aR1 genotype. However, cohousing of C5aR1 sufficient and deficient wildtype and Arctic mice resulted in a convergence of the microbiomes and equalized abundances of each identified order and genus across all genotypes. Similarly, pharmacologic treatment with the C5aR1 antagonist, PMX205, beginning at the onset of beta-amyloid plaque deposition in the Arctic and Tg2576 mice, demonstrated no impact of C5aR1 inhibition on the microbiome. This study demonstrates the importance of C1q in microbiota homeostasis in neurodegenerative disease. In addition, while demonstrating that constitutive deletion of C5aR1 can significantly alter the composition of the fecal microbiome, these differences are not present when C5aR1-deficient mice are cohoused with C5aR1-sufficient animals with or without the AD phenotype and suggests limited if any contribution of the microbiome to the previously observed prevention of cognitive and neuronal loss in the C5aR1-deficient AD models.

Helminths produce calreticulin (CRT) to immunomodulate the host immune system as a survival strategy. However, the structure of helminth-derived CRT and the structural basis of the immune evasion process remains unclarified. Previous study found that the tissue-dwelling helminth Trichinella spiralis produces calreticulin (TsCRT), which binds C1q to inhibit activation of the complement classical pathway. Here, we used x-ray crystallography to resolve the structure of truncated TsCRT (TsCRTΔ), the first structure of helminth-derived CRT. TsCRTΔ was observed to share the same binding region on C1q with IgG based on the structure and molecular docking, which explains the inhibitory effect of TsCRT on C1q-IgG-initiated classical complement activation. Based on the key residues in TsCRTΔ involved in the binding activity to C1q, a 24 amino acid peptide called PTsCRT was constructed that displayed strong C1q-binding activity and inhibited C1q-IgG-initiated classical complement activation. This study is the first to elucidate the structural basis of the role of TsCRT in immune evasion, providing an approach to develop helminth-derived bifunctional peptides as vaccine target to prevent parasite infections or as a therapeutic agent to treat complement-related autoimmune diseases. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) is a common finding in hospitalized patients, particularly those who are critically ill. The development of AKI is associated with several adverse outcomes including mortality, morbidity, progression to chronic kidney disease, and an increase in healthcare expenditure. Despite the well-established negative impact of AKI and rigorous efforts to better define, identify, and implement targeted therapies, the overall approach to the treatment of AKI continues to principally encompass supportive measures. This enduring challenge is primarily due to the heterogeneous nature of insults that activate many independent and overlapping molecular pathways. Consequently, it is evident that the identification of common mechanisms that mediate the pathogenesis of AKI, independent of etiology and engaged pathophysiological pathways, is of paramount importance and could lead to the identification of novel therapeutic targets. To better distinguish the commonly modulated mechanisms of AKI, we explored the transcriptional characteristics of human kidney biopsies from patients with acute tubular necrosis (ATN), and acute interstitial nephritis (AIN) using a NanoString inflammation panel. Subsequently, we used publicly available single-cell transcriptional resources to better interpret the generated transcriptional findings. Our findings identify robust acute kidney injury (AKI-induced) developmental reprogramming of macrophages (MΦ) with the expansion of C1Q+, CD163+ MΦ that is independent of the etiology of AKI and conserved across mouse and human species. These results would expand the current understanding of the pathophysiology of AKI and potentially offer novel targets for additional studies to enhance the translational transition of AKI research. NEW & NOTEWORTHY: Our findings identify robust acute kidney injury (AKI)-induced developmental reprogramming of macrophages (MΦ) with the expansion of C1Q+, CD163+ MΦ that is independent of the etiology of AKI and conserved across mouse and human species. [ABSTRACT FROM AUTHOR]

Cognitive impairment in learning, memory, and executive function occurs in normal aging even in the absence of Alzheimer's disease (AD). While neurons do not degenerate in humans or monkeys free of AD, there are structural changes including synapse loss and dendritic atrophy, especially in the dorsolateral prefrontal cortex (dlPFC), and these correlate with cognitive age-related impairment. Developmental studies revealed activity-dependent neuronal properties that lead to synapse remodeling by microglia. Microglia-mediated phagocytosis that may eliminate synapses is regulated by immune "eat me" and "don't eat me" signaling proteins in an activity-dependent manner, so that less active synapses are eliminated. Whether this process contributes to age-related synapse loss remains unknown. The present study used a rhesus monkey model of normal aging to investigate the balance between the "eat me" signal, complement component C1q, and the "don't eat me" signal, transmembrane glycoprotein CD47, relative to age-related synapse loss in dlPFC Area 46. Results showed an age-related elevation of C1q and reduction of CD47 at PSD95+ synapses that is associated with cognitive impairment. Additionally, reduced neuronal CD47 RNA expression was found, indicating that aged neurons were less able to produce the protective signal CD47. Interestingly, microglia do not show the hypertrophic morphology indicative of phagocytic activity. These findings suggest that in the aging brain, changes in the balance of immunologic proteins give microglia instructions favoring synapse elimination of less active synapses, but this may occur by a process other than classic phagocytosis such as trogocytosis. [ABSTRACT FROM AUTHOR]

Nephritis is a frequent and severe complication of Systemic Lupus Erythematous (SLE). The clinical course of lupus nephritis (LN) is usually characterized by alternating phases of remission and exacerbation. Flares of LN can lead to deterioration of kidney function, necessitating timely diagnosis and therapy. The presence of autoantibodies against C1q (anti-C1qAb) in the sera of SLE patients has been reported in various studies. Some research suggests that the presence and changes in the titer of anti-C1qAb may be associated with the development of LN, as well as with LN activity and renal flares. However, the exact role of anti-C1qAb in LN remains a subject of debate. Despite variability in the results of published studies, anti-C1qAb hold promise as noninvasive markers for assessing LN activity in SLE patients. Measuring anti-C1qAb levels could aid in diagnosing and managing LN during periods of both inactive disease and renal flares. Nevertheless, larger controlled trials with standardized laboratory assays are necessary to further establish the utility of anti-C1qAb in predicting the reactivation and remission of LN and guiding treatment strategies.

Endometriosis (EM) is defined as the engraftment and proliferation of functional endometrial-like tissue outside the uterine cavity, leading to a chronic inflammatory condition. While the precise etiology of EM remains elusive, recent studies have highlighted the crucial involvement of a dysregulated immune system. The complement system is one of the predominantly altered immune pathways in EM. Owing to its involvement in the process of angiogenesis, here, we have examined the possible role of the first recognition molecule of the complement classical pathway, C1q. C1q plays seminal roles in several physiological and pathological processes independent of complement activation, including tumor growth, placentation, wound healing, and angiogenesis. Gene expression analysis using the publicly available data revealed that C1q is expressed at higher levels in EM lesions compared to their healthy counterparts. Immunohistochemical analysis confirmed the presence of C1q protein, being localized around the blood vessels in the EM lesions. CD68+ macrophages are the likely producer of C1q in the EM lesions since cultured EM cells did not produce C1q in vitro. To explore the underlying reasons for increased C1q expression in EM, we focused on its established pro-angiogenic role. Employing various angiogenesis assays on primary endothelial endometriotic cells, such as migration, proliferation, and tube formation assays, we observed a robust proangiogenic effect induced by C1q on endothelial cells in the context of EM. C1q promoted angiogenesis in endothelial cells isolated from EM lesions (as well as healthy ovary that is also rich in C1q). Interestingly, endothelial cells from EM lesions seem to overexpress the receptor for the globular heads of C1q (gC1qR), a putative C1q receptor. Experiments with siRNA to silence gC1qR resulted in diminished capacity of C1q to perform its angiogenic functions, suggesting that C1q is likely to engage gC1qR in the pathophysiology of EM. gC1qR can be a potential therapeutic target in EM patients that will disrupt C1q-mediated proangiogenic activities in EM.

IntroductionSignificant neurologic morbidity is caused by pediatric cerebrospinal fluid (CSF) shunt infections. The underlying mechanisms leading to impaired school performance and increased risk of seizures are unknown, however, a better understanding of these mechanisms may allow us to temper their consequences. Recent evidence has demonstrated important roles for complement proteins in neurodevelopment and neuroinflammation.MethodsWe examined complement activation throughout Staphylococcus epidermidis (S. epidermidis) central nervous system (CNS) catheter infection. In addition, based on accumulating evidence that C3 plays a role in synaptic pruning in other neuroinflammatory states we determined if C3 and downstream C5 led to alterations in synaptic protein levels. Using our murine model of S. epidermidis catheter infection we quantified levels of the complement components C1q, Factor B, MASP2, C3, and C5 over the course of infection along with bacterial burdens.ResultsWe found that MASP2 predominated early in catheter infection, but that Factor B was elevated at intermediate time points. Unexpectedly C1q was elevated at late timepoints when bacterial burdens were low or undetectable. Based on these findings and the wealth of information regarding the emerging roles of C1q in the CNS, this suggests functions beyond pathogen elimination during S. epidermidis CNS catheter infection. To identify if C3 impacted synaptic protein levels we performed synaptosome isolation and quantified levels of VGLUT1 and PSD95 as well as pre-, post- and total synaptic puncta in cortical layer V of C3 knockout (KO) and wild type mice. We also used C5 KO and wild type mice to determine if there was any difference in pre-, post- and total synaptic puncta.DiscussionNeither C3 nor C5 impacted synaptic protein abundance. These findings suggest that chronic elevations in C1q in the brain that persist once CNS catheter infection has resolved may be modulating disease sequalae.

Objective To investigate the serum level of complement C1q in patients with malignant tumors and its clinical significance in the diagnosis of the tumors. Methods A retrospective analysis was conducted on 468 patients with malignant tumors (including 164 cases of lung malignant tumors, 123 cases of liver malignant tumors, 108 cases of gastric malignant tumors, and 73 cases of multiple myeloma) who were newly diagnosed and had not received any treatment measures from September 2020 to March 2021 in our hospital. Another 50 healthy individuals who taking physical examinations were subjected as control group. The serum level of complement C1q was quantitatively detected using the immunoturbidimetric assay. The levels were compared between the control and the patient groups. Results The serum C1q level was 155.17±34.81, 164.76±48.79, 156.87±32.70 and 124.39±31.71 mg/L, respectively in the lung, liver and gastric malignant tumor groups and multiple myeloma group, and the levels were significantly lower than that of the healthy control group (191.69±21.94 mg/L, P < 0.05). The sensitivity of serum C1q detection in the 4 malignant tumor groups was all above 90%, and the diagnostic efficacy of multiple myeloma was better (sensitivity 91.7%, specificity 86.3%). Using the normal group as a control, ROC curve analysis indicated that the cut-off value of serum complement C1q level in multiple myeloma patients was 157.8 mg/L, with an AUC area of 0.954; and the value was 164.3, 164.4, 164.3 and 164.4 mg/L for c lung, liver and gastric malignant tumors and all these tumors, respectively, with an AUC value of 0.836, 0.757, 0.817 and 0.830, respectively. Conclusion Serum C1q level determination has auxiliary diagnostic significance for malignant tumors, especially for the diagnosis of multiple myeloma.

The complement component C1q plays a role as a pro-angiogenic factor in different contexts, acting in a complement-independent way. For example, this molecule is able to foster the remodeling of the spiral arteries for a physiological pregnancy and to promote the wound healing process. It is also involved in angiogenesis after post-stroke ischemia. Furthermore, it has a role in supporting the tumor vessel growth. Given its role in promoting angiogenesis both under physiological and pathological situations, other studies are needed to understand its potential therapeutic implications.

Abstract Background Progression of Alzheimer’s disease leads to synapse loss, neural network dysfunction and cognitive failure. Accumulation of protein aggregates and brain immune activation have triggering roles in synaptic failure but the neuronal mechanisms underlying synapse loss are unclear. On the neuronal surface, cellular prion protein (PrPC) is known to be a high-affinity binding site for Amyloid-β oligomers (Aβo). However, PrPC’s dependence in knock-in AD models for tau accumulation, transcriptomic alterations and imaging biomarkers is unknown. Methods The necessity of PrPC was examined as a function of age in homozygous App NL−G−F /hMapt double knock-in mice (DKI). Phenotypes of App NL−G−F /hMapt mice with a deletion of Prnp expression (DKI; Prnp −/− ) were compared with DKI mice with intact Prnp, mice with a targeted deletion of Prnp (Prnp −/− ), and mice with intact Prnp (WT). Phenotypes examined included behavioral deficits, synapse loss by PET imaging, synapse loss by immunohistology, tau pathology, gliosis, inflammatory markers, and snRNA-seq transcriptomic profiling. Results By 9 months age, DKI mice showed learning and memory impairment, but DKI; Prnp −/− and Prnp −/− groups were indistinguishable from WT. Synapse loss in DKI brain, measured by [18F]SynVesT-1 SV2A PET or anti-SV2A immunohistology, was prevented by Prnp deletion. Accumulation of Tau phosphorylated at aa 217 and 202/205, C1q tagging of synapses, and dystrophic neurites were all increased in DKI mice but each decreased to WT levels with Prnp deletion. In contrast, astrogliosis, microgliosis and Aβ levels were unchanged between DKI and DKI; Prnp −/− groups. Single-nuclei transcriptomics revealed differential expression in neurons and glia of DKI mice relative to WT. For DKI; Prnp −/− mice, the majority of neuronal genes differentially expressed in DKI mice were no longer significantly altered relative to WT, but most glial DKI-dependent gene expression changes persisted. The DKI-dependent neuronal genes corrected by Prnp deletion associated bioinformatically with synaptic function. Additional genes were uniquely altered only in the Prnp −/− or the DKI; Prnp −/− groups. Conclusions Thus, PrPC-dependent synapse loss, phospho-tau accumulation and neuronal gene expression in AD mice can be reversed without clearing Aβ plaque or preventing gliotic reaction. This supports targeting the Aβo-PrPC interaction to prevent Aβo-neurotoxicity and pathologic tau accumulation in AD.

Feline infectious peritonitis (FIP) is a fatal disease in cats caused by infection with feline coronavirus (FCoV). Despite severe inflammatory changes, defense mechanisms fail to achieve virus clearance. Some studies focused on various immune evasion mechanisms, but none of these studies elucidated the inefficacy of the complement system, which is one major player in FIP-associated immune pathogenesis. This study aimed to investigate the involvement of complement-regulating factors (CRFs). CRFs help modulate the immune response and prevent host tissue damage. Archived tissue samples from 31 deceased, FIP-affected cats were evaluated using multiplex immunohistochemistry for the spatial expression of the complement-regulating factors CD46 and CD59 in association with FIP lesions and their colocalization with complement-activating factor C1q and membrane attack complex C9 in relation to the presence and proximity of FCoV-infected cells. The FIP lesions of all 31 cats exhibited marked expression of both complement-regulating factors in proximity to FCoV-infected macrophages. Moreover, their expression in all 31 animals was significantly lower than the expression of the complement-activating factors C1q and C9 compared to areas farther distal to FCoV-infected cells. In conclusion, FCoV-infected macrophages in cats with FIP appear to use autocrine and paracrine expression of complement-regulating factors in their immediate environment to shield themselves from destruction by the complement system.

Abstract Understanding the microglial neuro-immune interactions in the primate brain is vital to developing therapeutics for cortical injury, such as stroke or traumatic brain injury. Our previous work showed that mesenchymal-derived extracellular vesicles (MSC-EVs) enhanced motor recovery in aged rhesus monkeys following injury of primary motor cortex (M1), by promoting homeostatic ramified microglia, reducing injury-related neuronal hyperexcitability, and enhancing synaptic plasticity in perilesional cortices. A focal lesion was induced via surgical ablation of pial blood vessels over lying the cortical hand representation of M1 of aged female rhesus monkeys, that received intravenous infusions of either vehicle (veh) or EVs 24 h and again 14 days post-injury. The current study used this same cohort to address how these injury- and recovery-associated changes relate to structural and molecular interactions between microglia and neuronal synapses. Using multi-labeling immunohistochemistry, high-resolution microscopy, and gene expression analysis, we quantified co-expression of synaptic markers (VGLUTs, GLURs, VGAT, GABARs), microglia markers (Iba1, P2RY12), and C1q, a complement pathway protein for microglia-mediated synapse phagocytosis, in perilesional M1 and premotor cortices (PMC). We compared this lesion cohort to age-matched non-lesion controls (ctr). Our findings revealed a lesion-related loss of excitatory synapses in perilesional areas, which was ameliorated by EV treatment. Further, we found region-dependent effects of EVs on microglia and C1q expression. In perilesional M1, EV treatment and enhanced functional recovery were associated with increased expression of C1q + hypertrophic microglia, which are thought to have a role in debris-clearance and anti-inflammatory functions. In PMC, EV treatment was associated with decreased C1q + synaptic tagging and microglia–spine contacts. Our results suggest that EV treatment may enhance synaptic plasticity via clearance of acute damage in perilesional M1, and thereby preventing chronic inflammation and excessive synaptic loss in PMC. These mechanisms may act to preserve synaptic cortical motor networks and a balanced normative M1/PMC synaptic function to support functional recovery after injury.

Abstract The contribution of the gut microbiome to neuroinflammation, cognition, and Alzheimer’s disease progression has been highlighted over the past few years. Additionally, inhibition of various components of the complement system has repeatedly been demonstrated to reduce neuroinflammation and improve cognitive performance in AD mouse models. Whether the deletion of these complement components is associated with distinct microbiome composition, which could impact neuroinflammation and cognitive performance in mouse models has not yet been examined. Here, we provide a comprehensive analysis of conditional and constitutive knockouts, pharmacological inhibitors, and various housing paradigms for the animal models and wild-type controls at various ages. We aimed to determine the impact of C1q or C5aR1 inhibition on the microbiome in the Arctic and Tg2576 mouse models of AD, which develop amyloid plaques at different ages and locations. Analysis of fecal samples from WT and Arctic mice following global deletion of C1q demonstrated significant alterations to the microbiomes of Arctic but not WT mice, with substantial differences in abundances of Erysipelotrichales, Clostridiales and Alistipes. While no differences in microbiome diversity were detected between cohoused wildtype and Arctic mice with or without the constitutive deletion of the downstream complement receptor, C5aR1, a difference was detected between the C5aR1 sufficient (WT and Arctic) and deficient (C5ar1KO and ArcticC5aR1KO) mice, when the mice were housed segregated by C5aR1 genotype. However, cohousing of C5aR1 sufficient and deficient wildtype and Arctic mice resulted in a convergence of the microbiomes and equalized abundances of each identified order and genus across all genotypes. Similarly, pharmacologic treatment with the C5aR1 antagonist, PMX205, beginning at the onset of beta-amyloid plaque deposition in the Arctic and Tg2576 mice, demonstrated no impact of C5aR1 inhibition on the microbiome. This study demonstrates the importance of C1q in microbiota homeostasis in neurodegenerative disease. In addition, while demonstrating that constitutive deletion of C5aR1 can significantly alter the composition of the fecal microbiome, these differences are not present when C5aR1-deficient mice are cohoused with C5aR1-sufficient animals with or without the AD phenotype and suggests limited if any contribution of the microbiome to the previously observed prevention of cognitive and neuronal loss in the C5aR1-deficient AD models.

IntroductionLupus is a diverse autoimmune disease with autoantibody formation. Lupus nephritis carries a grave prognosis. Complement involvement, namely, C1q deficiency, is linked to activity and renal involvement and could help in their assessment. LN therapies include plasma exchange, immune adsorption, and probably hemodiafiltration with online endogenous reinfusion (HFR), together with traditional immunosuppressive therapies.AimThe aim of this study was to evaluate the role of HFR in improving signs and symptoms of systemic lupus erythematosus (SLE) activity and laboratory parameters in cases not responding to traditional immunosuppressive therapy.Settings and designA controlled clinical study was conducted on 60 patients with lupus from Group A that was subdivided into two groups: cases 1 (47 patients), those who received traditional medical treatment, and cases 2 (13 patients), those who underwent HFR in addition to medical treatment. Group B consisted of two subgroups: control 1, composed of 20 healthy age- and sex-matched volunteers, and control 2, consisting of 10 cases with different glomerular diseases other than lupus.Methods and materialsSerum C1q was determined before and after the HFR as well as induction by medical treatment. Disease activity was assessed using SLEDAI-2K with a responder index of 50; quality of life was assessed using SLEQOL v2, and HFR was performed for the non-responder group.ResultsC1q was lower in cases. It can efficiently differentiate between SLE patients and healthy controls with a sensitivity of 81.67% and a specificity of 90%. It can also efficiently differentiate between SLE patients and the control 2 group (non-lupus patients with renal glomerular disease) with a sensitivity of 83.33% and a specificity of 100%. C1q was more consumed in proliferative lupus, and correlated with anti-ds DNA, C3, and C4.ConclusionsC1q efficiently discriminates lupus patients and correlates with proliferative forms. HFR might ameliorate lupus activity and restore C1q.

Lack of complement factor C1q of the classical pathway results in severely impaired primary antibody responses. This is a paradox because antibodies, especially IgM, are the most efficient activators of the classical pathway and very little specific IgM will be present at priming. A possible explanation would be that natural IgM, binding with low affinity to the antigen, may suffice to activate complement. In support of this, mice lacking secretory IgM have an impaired antibody response,which can be rescued by transfer of non-immune IgM. Moreover, passive administration of specific IgM together with antigen enhances the antibody response in a complement-dependent fashion. To test the idea, we have used a knock-in mouse strain (Cm13) carrying a point mutation in the IgM heavy chain, rendering the IgM unable to activate complement. Mutant mice backcrossed to BALB/c or C57BL/6 background were primed and boosted with a low dose of sheep red blood cells. Confirming earlier data, no impairment in early, primary IgM- or IgG-responses were seen in either of the Cm13 strains. However, in one of the mutant strains, late primary IgG responses were impaired. A more pronounced effect was observed after boost, when the IgG response, the number of germinal center B cells and antibody secreting cells as well as the opsonization of antigen were impaired in mutant mice. We conclude that complement activation by natural IgM cannot explain the role of C1q in primary antibody responses, but that endogenous, specific, wildtype IgM generated after immunization feedback-enhances the response to a booster dose of antigen. Importantly, this mechanism can only partially explain the role of complement in the generation of antibody responses because the IgG response was much lower in C3- or complement receptor 1 and 2-deficient mice than in Cm13 mice. [ABSTRACT FROM AUTHOR]

Early identification of biomarkers that can predict the onset of sepsis-induced coagulopathy (SIC) in septic patients is clinically important. This study endeavors to examine the diagnostic and prognostic utility of serum C1q in the context of SIC. Clinical data from 279 patients diagnosed with sepsis at the Departments of Intensive Care, Respiratory Intensive Care, and Infectious Diseases at the Renmin Hospital of Wuhan University were gathered spanning from January 2022 to January 2024. These patients were categorized into two groups: the SIC group comprising 108 cases and the non-SIC group consisting of 171 cases, based on the presence of SIC. Within the SIC group, patients were further subdivided into a survival group (43 cases) and non-survival group (65 cases). The concentration of serum C1q in the SIC group was significantly lower than that in the non-SIC group. Furthermore, A significant correlation was observed between serum C1q levels and both SIC score and coagulation indices. C1q demonstrated superior diagnostic and prognostic performance for SIC patients, as indicated by a higher area under the curve (AUC). Notably, when combined with CRP, PCT, and SOFA score, C1q displayed the most robust diagnostic efficacy for SIC. Moreover, the combination of C1q with the SOFA score heightened predictive value concerning the 28-day mortality of SIC patients. [ABSTRACT FROM AUTHOR]

Background: Antibody-mediated complement fixation has previously been associated with protection against malaria in naturally acquired immunity. However, the process of early-life development of complement-fixing antibodies in infants, both in comparison to their respective mothers and to other immune parameters, remains less clear. Results: We measured complement-fixing antibodies in newborns and their mothers in a malaria endemic area over 5 years follow-up and found that infants’ complement-fixing antibody levels were highest at birth, decreased until six months, then increased progressively until they were similar to birth at five years. Infants with high levels at birth experienced a faster decay of complement-fixing antibodies but showed similar levels to the low response group of newborns thereafter. No difference was observed in antibody levels between infant cord blood and mothers at delivery. The same result was found when categorized into high and low response groups, indicating placental transfer of antibodies. Complement-fixing antibodies were positively correlated with total schizont-specific IgG and IgM levels in mothers and infants at several time points. At nine months, complement-fixing antibodies were negatively correlated with total B cell frequency and osteopontin concentrations in the infants, while positively correlated with atypical memory B cells and P. falciparum-positive atypical memory B cells. Conclusion: This study indicates that complement-fixing antibodies against P. falciparum merozoites are produced in the mothers and placentally-transferred, and they are acquired in infants over time during the first years of life. Understanding early life immune responses is crucial for developing a functional, long lasting malaria vaccine. [ABSTRACT FROM AUTHOR]

Background: Progression of Alzheimer's disease leads to synapse loss, neural network dysfunction and cognitive failure. Accumulation of protein aggregates and brain immune activation have triggering roles in synaptic failure but the neuronal mechanisms underlying synapse loss are unclear. On the neuronal surface, cellular prion protein (PrPC) is known to be a high-affinity binding site for Amyloid-β oligomers (Aβo). However, PrPC's dependence in knock-in AD models for tau accumulation, transcriptomic alterations and imaging biomarkers is unknown. Methods: The necessity of PrPC was examined as a function of age in homozygous AppNL−G−F/hMapt double knock-in mice (DKI). Phenotypes of AppNL−G−F/hMapt mice with a deletion of Prnp expression (DKI; Prnp−/−) were compared with DKI mice with intact Prnp, mice with a targeted deletion of Prnp (Prnp−/−), and mice with intact Prnp (WT). Phenotypes examined included behavioral deficits, synapse loss by PET imaging, synapse loss by immunohistology, tau pathology, gliosis, inflammatory markers, and snRNA-seq transcriptomic profiling. Results: By 9 months age, DKI mice showed learning and memory impairment, but DKI; Prnp−/− and Prnp−/− groups were indistinguishable from WT. Synapse loss in DKI brain, measured by [18F]SynVesT-1 SV2A PET or anti-SV2A immunohistology, was prevented by Prnp deletion. Accumulation of Tau phosphorylated at aa 217 and 202/205, C1q tagging of synapses, and dystrophic neurites were all increased in DKI mice but each decreased to WT levels with Prnp deletion. In contrast, astrogliosis, microgliosis and Aβ levels were unchanged between DKI and DKI; Prnp−/− groups. Single-nuclei transcriptomics revealed differential expression in neurons and glia of DKI mice relative to WT. For DKI; Prnp−/− mice, the majority of neuronal genes differentially expressed in DKI mice were no longer significantly altered relative to WT, but most glial DKI-dependent gene expression changes persisted. The DKI-dependent neuronal genes corrected by Prnp deletion associated bioinformatically with synaptic function. Additional genes were uniquely altered only in the Prnp−/− or the DKI; Prnp−/− groups. Conclusions: Thus, PrPC-dependent synapse loss, phospho-tau accumulation and neuronal gene expression in AD mice can be reversed without clearing Aβ plaque or preventing gliotic reaction. This supports targeting the Aβo-PrPC interaction to prevent Aβo-neurotoxicity and pathologic tau accumulation in AD. [ABSTRACT FROM AUTHOR]

The interaction between IgM and C1q represents the first step of the classical pathway of the complement system in higher vertebrates. To identify the significance of particular IgM/C1q interactions, recombinant IgMs were used in both hexameric and pentameric configurations and with two different specificities, along with C1q derived from human serum (sC1q) and two recombinant single-chain variants of the trimeric globular region of C1q. Interaction and complement activation assays were performed using the ELISA format, and bio-layer interferometry measurements to study kinetic behavior. The differences between hexameric and pentameric IgM conformations were only slightly visible in the interaction assay, but significant in the complement activation assay. Hexameric IgM requires a lower concentration of sC1q to activate the complement compared to pentameric IgM, leading to an increased release of C4 compared to pentameric IgM. The recombinant C1q mimetics competed with sC1q in interaction assays and were able to inhibit complement activation. The bio-layer interferometry measurements revealed KD values in the nanomolar range for the IgM/C1q interaction, while the C1q mimetics exhibited rapid on and off binding rates with the IgMs. Our results make C1q mimetics valuable tools for developing recombinant C1q, specifically its variants, for further scientific studies and clinical applications.

Lack of complement factor C1q of the classical pathway results in severely impaired primary antibody responses. This is a paradox because antibodies, especially IgM, are the most efficient activators of the classical pathway and very little specific IgM will be present at priming. A possible explanation would be that natural IgM, binding with low affinity to the antigen, may suffice to activate complement. In support of this, mice lacking secretory IgM have an impaired antibody response, which can be rescued by transfer of non-immune IgM. Moreover, passive administration of specific IgM together with antigen enhances the antibody response in a complement-dependent fashion. To test the idea, we have used a knock-in mouse strain (Cμ13) carrying a point mutation in the IgM heavy chain, rendering the IgM unable to activate complement. Mutant mice backcrossed to BALB/c or C57BL/6 background were primed and boosted with a low dose of sheep red blood cells. Confirming earlier data, no impairment in early, primary IgM- or IgG-responses were seen in either of the Cμ13 strains. However, in one of the mutant strains, late primary IgG responses were impaired. A more pronounced effect was observed after boost, when the IgG response, the number of germinal center B cells and antibody secreting cells as well as the opsonization of antigen were impaired in mutant mice. We conclude that complement activation by natural IgM cannot explain the role of C1q in primary antibody responses, but that endogenous, specific, wildtype IgM generated after immunization feedback-enhances the response to a booster dose of antigen. Importantly, this mechanism can only partially explain the role of complement in the generation of antibody responses because the IgG response was much lower in C3- or complement receptor 1 and 2-deficient mice than in Cμ13 mice.

Atherosclerosis is an inflammatory disease of the vessel wall, with cholesterol crystal (CC) deposition being a hallmark of the disease. As evidence for a cross-talk between complement activation and hemostasis on CC surfaces has been limited to in vitro data, the aim of this study was to demonstrate the presence of C1q-vWF complexes in human atherosclerosis ex vivo. We used immunofluorescence staining and a proximity ligation assay (PLA, Duolink®) to examine the presence, localization, and co-localization of C1q and vWF in frozen sections of human carotid arteries with atherosclerosis or without atherosclerotic changes as well as material from thrombendarteriectomy. We observed significantly higher levels of C1q and vWF in healthy tissue compared to diseased material and greater co-localization in the PLA in healthy samples than in diseased samples. In diseased samples, fluorescence signals were highest in locations encompassing atheroma and foam cells. While there was overall reduced signal in areas with CCs, the staining was spotty, and there was evidence of co-localization on individual CCs. Thus, we demonstrate the presence of C1q-vWF complexes in human carotid arteries ex vivo, which was most abundant in healthy endothelial and subendothelial space and reduced in diseased tissue. C1q-vWF interaction can also be demonstrated on the CC surface.

ABSTRACTStreptococcus equi subsp. zooepidemicus (SEZ) is a major equine pathogen that causes pneumonia, abortion, and polyarthritis. It can also cause invasive infections in humans. SEZ expresses the M-like protein SzM, which recruits host proteins such as fibrinogen to the bacterial surface. Equine SEZ strain C2, which binds only comparably low amounts of human fibrinogen in comparison to human SEZ strain C33, was previously shown to proliferate in equine and human blood. As the expression of SzM_C2 was necessary for survival in blood, this study investigated the working hypothesis that SzM_C2 inhibits complement activation through a mechanism other than fibrinogen and non-immune immunoglobulin binding. Loss-of-function experiments showed that SEZ C2, but not C33, binds C1q via SzM in IgG-free human plasma. Furthermore, SzM C2 expression is necessary for recruiting purified human or equine C1q to the bacterial surface. Flow cytometry analysis demonstrated that SzM expression in SEZ C2 is crucial for the significant reduction of C3b labelling in human plasma. Addition of human plasma to immobilized rSzM_C2 and immobilized aggregated IgG led to binding of C1q, but only the latter activated the complement system, as shown by the detection of C4 deposition. Complement activation induced by aggregated IgG was significantly reduced if human plasma was pre-incubated with rSzM_C2. Furthermore, rSzM_C2, but not rSzM_C33, inhibited the activation of the classical complement pathway in human plasma, as determined in an erythrocyte lysis experiment. In conclusion, the immunoglobulin-independent binding of C1q to SzM_C2 is associated with complement inhibition.