Cells die by necrosis due to excessive chemical or thermal stress, leading to plasma membrane rupture, release of intracellular components and severe inflammation. The clearance of necrotic cell debris is crucial for tissue recovery and injury resolution, however, the underlying mechanisms are still poorly understood, especially in vivo. This study examined the role of complement proteins in promoting clearance of necrotic cell debris by leukocytes and their influence on liver regeneration. We found that independently of the type of necrotic liver injury, either paracetamol (APAP) overdose or thermal injury, complement proteins C1q and (i)C3b were deposited specifically on necrotic lesions via the activation of the classical pathway. Importantly, C3 deficiency led to a significant accumulation of necrotic debris and impairment of liver recovery in mice, which was attributed to decreased phagocytosis of debris by recruited neutrophils in vivo. Monocytes and macrophages also took part in debris clearance, although the necessity of C3 and CD11b was dependent on the specific type of necrotic liver injury. Using human neutrophils, we showed that depletion of C1q or C3 caused a reduction in the volume of necrotic debris that is phagocytosed, indicating that complement promotes effective debris uptake by neutrophils in mice and humans. In summary, complement activation at injury sites is a pivotal event for necrotic debris clearance by phagocytes and determinant for efficient recovery from tissue injury. Abstract Figure Key points The complement cascade is activated on necrotic cell debris in vivo via the classical pathway Deficiency in complement C3 impairs necrotic debris clearance and liver recovery after injury Complement-mediated debris clearance is performed by neutrophils, monocytes and macrophages Human neutrophils depend on complement opsonization to phagocytose necrotic cell debris efficiently

Systemic candidiasis is a common, high-mortality, nosocomial fungal infection. Unexpectedly, it has emerged as a complication of anti-complement C5-targeted monoclonal antibody treatment, indicating a critical niche for C5 in antifungal immunity. We identified transcription of complement system genes as the top biological pathway induced in candidemic patients and as predictive of candidemia. Mechanistically, C5a-C5aR1 promoted fungal clearance and host survival in a mouse model of systemic candidiasis by stimulating phagocyte effector function and ERK- and AKT-dependent survival in infected tissues. C5ar1 ablation rewired macrophage metabolism downstream of mTOR, promoting their apoptosis and enhancing mortality through kidney injury. Besides hepatocyte-derived C5, local C5 produced intrinsically by phagocytes provided a key substrate for antifungal protection. Lower serum C5a concentrations or a C5 polymorphism that decreases leukocyte C5 expression correlated independently with poor patient outcomes. Thus, local, phagocyte-derived C5 production licenses phagocyte antimicrobial function and confers innate protection during systemic fungal infection.
Published by Elsevier Inc.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Approximately one-third of MB patients remain incurable. Understanding the molecular mechanism of MB tumorigenesis is, therefore, critical for developing specific and effective treatment strategies. Our previous work demonstrated that astrocytes constitute the tumor microenvironment (TME) of MB and play an indispensable role in MB progression. However, the underlying mechanisms by which astrocytes are regulated and activated to promote MB remain elusive.
By taking advantage of Math1-Cre/Ptch1loxp/loxp mice, which spontaneously develop MB, primary MB cells and astrocytes were isolated and then subjected to administration and coculture in vitro. Immunohistochemistry was utilized to determine the presence of C3a in MB sections. MB cell proliferation was evaluated by immunofluorescent staining. GFAP and cytokine expression levels in C3a-stimulated astrocytes were assessed by immunofluorescent staining, western blotting, q-PCR and ELISA. C3a receptor and TNF-α receptor expression was determined by PCR and immunofluorescent staining. p38 MAPK pathway activation was detected by western blotting. Transplanted MB mice were treated with a C3a receptor antagonist or TNF-α receptor antagonist to investigate their role in MB progression in vivo.
We found that complement C3a, a fragment released from intact complement C3 following complement activation, was enriched in both human and murine MB tumor tissue, and its receptor was highly expressed on tumor-associated astrocytes (TAAs). We demonstrated that C3a activated astrocytes and promoted MB cell proliferation via the p38 MAPK pathway. Moreover, we discovered that C3a upregulated the production of proinflammatory cytokines, such as IL-6 and TNF-α in astrocytes. Application of the conditioned medium of C3a-stimulated astrocytes promoted MB cell proliferation, which was abolished by preincubation with a TNF-α receptor antagonist, indicating a TNF-α-dependent event. Indeed, we further demonstrated that administration of a selective C3a receptor or TNF-α receptor antagonist to mice subcutaneously transplanted with MB suppressed tumor progression in vivo.
C3a was released during MB development. C3a triggered astrocyte activation and TNF-α production via the p38 pathway, which promoted MB cell proliferation. Our findings revealed the novel role of C3a-mediated TNF-α production by astrocytes in MB progression. These findings imply that targeting C3a and TNF-α may represent a potential novel therapeutic approach for human MB.
© 2022. The Author(s).

Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, causes personal suffering and high socioeconomic costs. While there has been progress in the treatments for the neovascular form of AMD, no therapy is yet available for the more common dry form, also known as geographic atrophy. We analysed the retinal tissue in a mouse model of retinal degeneration caused by sodium iodate (NaIO3)-induced retinal pigment epithelium (RPE) atrophy to understand the underlying pathology. RNA sequencing (RNA-seq), qRT-PCR, Western blot, immunohistochemistry of the retinas and multiplex ELISA of the mouse serum were applied to find the pathways involved in the degeneration. NaIO3 caused patchy RPE loss and thinning of the photoreceptor layer. This was accompanied by the increased retinal expression of complement components c1s, c3, c4, cfb and cfh. C1s, C3, CFH and CFB were complement proteins, with enhanced deposition at day 3. C4 was upregulated in retinal degeneration at day 10. Consistently, the transcript levels of proinflammatory ccl-2, -3, -5, il-1β, il-33 and tgf-β were increased in the retinas of NaIO3 mice, but vegf-a mRNA was reduced. Macrophages, microglia and gliotic Müller cells could be a cellular source for local retinal inflammatory changes in the NaIO3 retina. Systemic complement and cytokines/chemokines remained unaltered in this model of NaIO3-dependent retinal degeneration. In conclusion, systemically administered NaIO3 promotes degenerative and inflammatory processes in the retina, which can mimic the hallmarks of geographic atrophy.

Myocarditis can lead to autoimmune disease, dilated cardiomyopathy, and heart failure, which is modeled in the mouse by cardiac myosin immunization (experimental autoimmune myocarditis [EAM]). Signal transducer and activator of transcription 3 (STAT3) systemic inhibition exerts both preventive and therapeutic effects in EAM, and STAT3 constitutive activation elicits immune-mediated myocarditis dependent on complement C3 and correlating with activation of the STAT3-interleukin 6 (IL-6) axis in the liver. Thus, liver-specific STAT3 inhibition may represent a therapeutic option, allowing to bypass the heart toxicity, predicted by systemic STAT3 inhibition. We therefore decided to explore the effectiveness of silencing liver Stat3 and C3 in preventing EAM onset and/or the recovery of cardiac functions. We first show that complement C3 and C5 genetic depletion significantly prevents the onset of spontaneous myocarditis, supporting the complement cascade as a viable target. In order to interfere with complement production and STAT3 activity specifically in the liver, we took advantage of liver-specific Stat3 or C3 small interfering (si)RNA nanoparticles, demonstrating that both siRNAs can significantly prevent myocarditis onset and improve the recovery of heart functions in EAM. Our data demonstrate that liver-specific Stat3/C3 siRNAs may represent a therapeutic option for autoimmune myocarditis and suggest that complement levels and activation might be predictive of progression to dilated cardiomyopathy.
© 2020 The Author(s).