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.

Macular fibrosis causes irreparable vision loss in neovascular age-related macular degeneration (nAMD) even with anti-vascular endothelial growth factor (VEGF) therapy. Inflammation is known to play an important role in macular fibrosis although the underlying mechanism remains poorly defined. The aim of this study was to understand how infiltrating macrophages and complement proteins may contribute to macular fibrosis.
Subretinal fibrosis was induced in C57BL/6J mice using the two-stage laser protocol developed by our group. The eyes were collected at 10, 20, 30 and 40 days after the second laser and processed for immunohistochemistry for infiltrating macrophages (F4/80 and Iba-1), complement components (C3a and C3aR) and fibrovascular lesions (collagen-1, Isolectin B4 and α-SMA). Human retinal sections with macular fibrosis were also used in the study. Bone marrow-derived macrophages (BMDMs) from C57BL/6J mice were treated with recombinant C3a, C5a or TGF-β for 48 and 96 h. qPCR, Western blot and immunohistochemistry were used to examine the expression of myofibroblast markers. The involvement of C3a-C3aR pathway in macrophage to myofibroblast transition (MMT) and subretinal fibrosis was further investigated using a C3aR antagonist (C3aRA) and a C3a blocking antibody in vitro and in vivo.
Approximately 20~30% of F4/80+ (or Iba-1+) infiltrating macrophages co-expressed α-SMA in subretinal fibrotic lesions both in human nAMD eyes and in the mouse model. TGF-β and C3a, but not C5a treatment, significantly upregulated expression of α-SMA, fibronectin and collagen-1 in BMDMs. C3a-induced upregulation of α-SMA, fibronectin and collagen-1 in BMDMs was prevented by C3aRA treatment. In the two-stage laser model of induced subretinal fibrosis, treatment with C3a blocking antibody but not C3aRA significantly reduced vascular leakage and Isolectin B4+ lesions. The treatment did not significantly alter collagen-1+ fibrotic lesions.
MMT plays a role in macular fibrosis secondary to nAMD. MMT can be induced by TGF-β and C3a but not C5a. Further research is required to fully understand the role of MMT in macular fibrosis. Macrophage to myofibroblast transition (MMT) contributes to subretinal fibrosis. Subretinal fibrosis lesions contain various cell types, including macrophages and myofibroblasts, and are fibrovascular. Myofibroblasts are key cells driving pathogenic fibrosis, and they do so by producing excessive amount of extracellular matrix proteins. We have found that infiltrating macrophages can transdifferentiate into myofibroblasts, a phenomenon termed macrophage to myofibroblast transition (MMT) in macular fibrosis. In addition to TGF-β1, C3a generated during complement activation in CNV can also induce MMT contributing to macular fibrosis. RPE = retinal pigment epithelium. BM = Bruch's membrane. MMT = macrophage to myofibroblast transition. TGFB = transforming growth factor β. a-SMA = alpha smooth muscle actin. C3a = complement C3a.