Cortical demyelination and neurodegeneration are associated with a more severe and disabling multiple sclerosis (MS) (Calabrese et al., 2015; Absinta et al., 2020). Infiltrates of immune cells in the leptomeningeal and perivascular spaces, which are a feature of the broader compartmentalised inflammatory response that characterises established MS (Lassmann, 2019), associate with the extent of cortical pathology (Magliozzi et al., 2007; Reynolds et al., 2011; Lassmann, 2019; Kee et al., 2022). These cellular infiltrates represent an intrathecal source of soluble cytokines and inflammatory signals, which may activate microglia and astrocytes, so they become damaging to myelin or directly cytotoxic to neurons in the subpial and perivascular tissues (Gardner et al., 2013; Magliozzi et al., 2019b). Recognising the biological pathways associated with cortical pathology and compartmentalised inflammation will be important to guide future attempts to target such clinically meaningful aspects of MS pathology.

Inflammation of the leptomeninges and perivascular space is associated with gradients of tissue injury, detectable on advanced MRI and at autopsy, that represent substantial neuronal and glial alterations, and are most pronounced in the subpial tissue but extend through the depth of the cortical grey matter (GM) (Peterson et al., 2001; Magliozzi et al., 2010; Mainero et al., 2015; Griffiths et al., 2020; Junker et al., 2020). A transcriptomic and biochemical signature of inflammation, enriched for cytokines that support tissue-homing and lymphoid neogenesis, alongside complement proteins, characterise the cerebrospinal fluid (CSF) of cases with more extensive cortical GM lesions at diagnosis. A finding replicated in cases characterised by extensive cortical demyelination and leptomeningeal inflammation at post-mortem (Magliozzi et al., 2018, 2019a,b, 2020). Complement proteins (including C1q, C3), activation products (C4d) and their receptors, including anaphylatoxin receptors (C3aR1 and C5aR1), are located at subpial, leukocortical, white matter (WM) and deep GM areas in MS, where they associate with the extent of myelin and neuronal loss (Breij et al., 2008; Barnett et al., 2009; Ingram et al., 2014; Michailidou et al., 2015, 2017; Watkins et al., 2016; Loveless et al., 2018; Cooze et al., 2022). The concentration of complement proteins in the CSF differs between MS and non-inflammatory controls and between MS subtypes, disease severity and outcome (Aeinehband et al., 2015; Håkansson et al., 2020). Complement and complement receptors are essential to B: T-cell co-stimulation, monocyte: lymphocyte interactions and lymphocyte chemotaxis (West et al., 2018). Complement activation at the blood-CSF and CSF-brain barriers of the choroid epithelium, ependyma, pia mater and vascular endothelia are a feature of neuromyelitis optica, and complement activation is noted at the choroid plexus and in acute WM lesions in MS (Compston et al., 1989; Wayne Moore et al., 2016; Guo et al., 2017). It is not clear whether raised complement proteins in the CSF are accompanied by complement activation at CSF—blood brain barriers of the leptomeninges, pia and vasculature, or if complement activation at these brain barriers is associated with inflammatory changes in the underlying cortical GM.

Complement activation results in generation of anaphylatoxins C3a and C5a, which bind to G-protein-coupled anaphylatoxin receptors C3aR1 and C5aR1 (CD88) expressed by monocytes, macrophages, microglia, and some activated astrocytes (Müller-Ladner et al., 1996; Gasque et al., 1997, 1998; Ischenko et al., 1998). Complement anaphylatoxins are raised in MS CSF (Ingram et al., 2010; Håkansson et al., 2020) and CSF C5a levels and C5a inhibition in the CSF ameliorates blood-brain barrier disruption (Faustmann et al., 1995; Flierl et al., 2009). Activation of C3aR1 and C5aR1 on immune cells promote upregulation of functional pathways relevant to inflammation (including oxidative burst), the release of soluble chemoattractant and pro-inflammatory cytokines [including tumour necrosis factor α (TNFα) and interferon γ] and stimulates phagocytosis and metabolic dysfunction (Haas and van Strijp, 2007; Klos et al., 2009; Vandendriessche et al., 2021). The extent of experimentally induced demyelination and inflammation is enhanced in mice overexpressing C3a and C5a, and deletion of C5aR1 or the application of C5aR1 antagonists, reduces the severity of injury (Nataf et al., 1998, 1999; Ingersoll et al., 2010; Peterson et al., 2017). These findings indicate the potential value of targeting the C5a—C5aR1 axis to improve disease outcome.

To better understand the contribution of complement activation and anaphylatoxin receptor expressing cells to processes of compartmentalised inflammation and cortical pathology, our post-mortem study of MS investigated: (i) the presence of products of complement activation at the brain surfaces (pia and around blood vessels of the cortex); (ii) the association between complement, microglial and astroglial activation and; (iii) differences in complement C3aR1 and C5aR1 positive cell numbers in areas of cortical demyelination in comparison to non-lesion and control GM samples. We found that complement at the brain surfaces associated with the extent of compartmentalised inflammation, cortical demyelination and complement anaphylatoxin receptor positive microglia at the expanding lesion edge. Collectively, our findings add to the weight of evidence implicating a key role for complement activation at the brain surface and complement-microglia interactions via C5aR1 to the pathological severity of MS.

We describe the presence of products of complement activation in the leptomeninges, perivascular spaces and parenchyma of MS characterised by extensive cortical demyelination and compartmentalised inflammation. The presence of complement activation proteins from both classical and alternative pathways implies complement activation at the CSF-brain barriers is ongoing throughout the disease process, which was in excess of 30 years in some cases. The localisation of complement activation proteins on damaged neurons, myelin and synapses is well described in the MS literature, but the relationship between complement activation at the brain barriers and changes in parenchymal, resident cell populations, such as microglia/macrophages, and the severity of GM and WM pathology is not clearly defined. Complement anaphylatoxin receptor positive cells (C3aR1 and C5aR1) were increased in density at, or near, cortical demyelinating lesions. C5aR1+ microglia/macrophages associated with ongoing expansion of both subpial and leukocortical GM lesions, displayed an altered morphology, expressed TNFα, iNOS and contained puncta immunoreactive for PLP, neurofilament and synaptophysin antigens. The generation of anaphylatoxin peptides associated with activated, proinflammatory microglia/macrophages and ongoing phagocytosis of neural and oligodendrocyte membranes suggests the C5a/C5aR1 axis may represent a treatment target to abrogate compartmentalised inflammation and cortical pathology in progressive MS.

Compartmentalised inflammation drives disease worsening independently of acute clinical relapses and inflammatory lesions (Cree et al., 2019). The compartmentalised inflammatory response is composed of activated immune cells in the parenchyma, reactive microglia and astroglia, increased numbers of leptomeningeal and connective tissue T and B lymphocytes, long-lived plasma cells, and elevated levels of circulating cytokines in the CSF (Lassmann, 2019). An overtly inflammatory CSF cytokine profile is associated with a more severe MS at presentation and follow-up, and pathologically, is linked to subpial tissue damage in a gradient-like pattern, which can be replicated in experimental models by the induced expression of inflammatory cytokines in the leptomeninges (Magliozzi et al., 2010, 2018, 2020; Gardner et al., 2013; James Bates et al., 2022). The levels of complement proteins, including C1q, C2, C4, and C5 in MS CSF and serum are altered in comparison to healthy individuals and between different MS clinical subtypes (Sellebjerg et al., 1998; Jongen et al., 2000; Finehout et al., 2005; Stoop et al., 2008; Ingram et al., 2010; Aeinehband et al., 2015; Magliozzi et al., 2019a; Zelek et al., 2019). Complement, together with serum proteins, including fibrin, represent effector proinflammatory molecules with the potential to signal across the CSF- brain barriers to activate microglia and astrocytes (Faustmann et al., 1995; Davalos et al., 2012; Ryu et al., 2015; Lee et al., 2018). Only a small number of prior studies have investigated the presence of complement proteins and activation products at the key CSF- and blood- brain barrier sites of pathological samples of MS (Wayne Moore et al., 2016; Guo et al., 2017). We show the widespread localisation of C1q, C4d, Bb, and C3b at the choroid plexus, ventricular ependyma and a relative increase at leptomeningeal and parenchymal vessels, which associated with the extent of cortical GM pathology. Therefore, complement deposition and activation at the pia and perivascular sites represents a component of the compartmentalised inflammatory response in MS, which is positioned to stimulate inflammation, lead to disruption of essential brain barriers, and support the formation or expansion of GM lesions by triggering local microglia and astrocytes.

The relative extent of complement immunoreactivity correlated modestly with the degree of leptomeningeal cellular infiltration and the extent of cortical GM demyelination (Figure 2), whilst parenchymal C3b immunoreactivity has been associated with activated microglia in the medial thalamus, another structure which lies close to CSF-filled spaces in the brain (Cooze et al., 2022). Infiltrating lymphocytes are a source of complement proteins in the subarachnoid space (Stahel et al., 1997) and the density of leptomeningeal infiltrates correlates closely with the degree of inflammation, demyelination, and neuro-axonal loss in the MS neocortex, cerebellum, and spinal cord (Magliozzi et al., 2007; Howell et al., 2011, 2015; Reali et al., 2020). The intrathecal activation of the classical (C1q, C4d) and alternative (Bb) pathways generates C3- and C5-convertases essential for the synthesis of the potent soluble anaphylatoxin fragments, which are also elevated in MS CSF (Ingram et al., 2010; Håkansson et al., 2020). The soluble complement anaphylatoxins can activate chemotactic, protective and neuron damaging functional pathways in microglia/macrophages and other cells by engaging with C3aR1 and C5aR1 (Woodruff et al., 2006; Ingersoll et al., 2010; Pavlovski et al., 2012). In addition, C3b-iC3b on cell surfaces is the ligand for complement receptor 3 (CR3, CD11b, MAC-1)-mediated phagocytosis and is important in mediating processes of synapse removal during development and in disease (Salter and Stevens, 2017). The phagocytosis of neural membranes and synapses labelled with C1q-C3 by activated microglia is seen in MS and its models, where reducing complement activation or increasing its clearance, protects neurites and synapses and preserves neurological function (Hammond et al., 2020; Werneburg et al., 2020; Gharagozloo et al., 2021; Ramaglia et al., 2021). Therefore, complement activation in the vicinity of resident microglial could be an important component mediating the extensive damage and loss of synapses, neurites and neurons of the cortical GM, which is a key component to the irreversible clinical worsening of progressive MS (Peterson et al., 2001; Magliozzi et al., 2010; Jürgens et al., 2016).

Several studies have documented the increased number of C3aR1 and C5aR1 cells in MS lesions (Gasque et al., 1998; Ischenko et al., 1998; Ingram et al., 2014), including cortical GM lesions (Watkins et al., 2016; Loveless et al., 2018). However, the identity of the cells most widely expressing the anaphylatoxin receptors, or their relative contribution to the extent of inflammation and demyelination, has not been clarified. Alongside a robust expression on myeloid lineage cells (Klos et al., 2013), earlier reports demonstrated anaphylatoxin receptor expression (both C3aR1 and C5aR1) on neurons, astrocytes and endothelia (Gasque et al., 1997, 1998; Ischenko et al., 1998; Davoust et al., 1999; Stahel et al., 2000; Farkas et al., 2003; Brennan et al., 2015). In MS, the expression appears restricted to myeloid cells, including microglia, and activated astrocytes (Müller-Ladner et al., 1996; Ischenko et al., 1998). We confirmed C3aR1 and C5aR1 expression by HLA-D+ and TMEM119 microglia/macrophages. Note that TMEM119 expression is downregulated in activated, non-homeostatic microglia, such as those found at the chronic active lesion edge (Zrzavy et al., 2017; Vankriekelsvenne et al., 2022). We did not anaphylatoxin receptor expression on cortical astrocytes, cells that are phenotypically and morphologically diverse and distinct from astrocytes of the WM (Oberheim et al., 2009; Schirmer et al., 2019). We confirmed a specific microglia/macrophage pattern of C3aR1 and C5aR1 expression in primary mouse dissociated cultures and induced human microglia-like cells. Induced human microglia-like cells expressed TMEM119 and TREM2 and are a useful source of human-derived cells for the analysis of microglial/macrophage biology (Sellgren et al., 2019). Microglia expressing TREM2 are associated with phagocytosis of apoptotic neurons and the subsequent loss of homeostatic function and the adoption of a neurodegenerative phenotype, which associated with neuronal loss (Krasemann et al., 2017). Reducing TREM2 expression on microglia attenuates inflammation and is protective against neurodegenerative pathology (Leyns et al., 2017). Therefore, our demonstration of the expression of TREM2, TMEM119 together with C3aR/C5aR1, is consistent with a population of reactive microglia that have lost some homeostatic functions and acquired detrimental phenotypic features.

It is important to note that complement can elicit a range of protective as well as damaging responses in the CNS. For example, C3a/C3aR1 and C5a/C5aR1 axis activation is reported to protect cultured neurons from excitotoxic death (van Beek et al., 2001; Mukherjee et al., 2008) and is associated with the induced release of neurotrophins, cell survival and enhanced neurogenesis, in some settings (Jauneau et al., 2006; Haynes et al., 2013; Westacott et al., 2021); whilst C3a and C5a overexpressing mice displayed similar rates of remyelination (Ingersoll et al., 2010), although presenting larger and more inflammatory demyelinating lesions. More work clearly needs to be done to fully understand the role of C3aR1 and C5aR1 activation at all stages of MS. A third receptor C5aR2 (C5L2 or GPR77), is less well characterised and regarded as a scavenger or default receptor competing with C5aR for C5a binding. The functional role for C5aR2 and its relationship to the intracellular signalling pathways regulating inflammation remains to be determined (Ward, 2009; Klos et al., 2013); expression of C5aR2 was not investigated in this study.

The borders of leukocortical and subpial GM lesions were demarcated by elevated numbers of HLA-D and CD68 + cells, which were mirrored by an increased number of C3aR1 and C5aR1+ microglia/macrophages. Previously, we have shown that the number of C1q and Bb immunopositive cells in MS leukocortical lesions correlated with the density of HLA-D+ and C5aR1+ microglia (Watkins et al., 2016). Our current findings implicate circulating as well as locally generated complement, to be playing a role in driving pathology in both subpial and deeper cortical laminae. C5aR1+ microglia within a chronic active GM lesion demonstrated subtle morphological alterations consistent with a transition of process bearing microglia to a more rounded and simpler morphological state (Figure 5), reminiscent of IBA-1+ microglia that associate with a more profound cortical pathology in progressive MS (van Olst et al., 2021). C5aR1+ microglia/macrophages in MS cortical GM co-stained for iNOS and TNFα and demonstrated cytoplasmic PLP, neurofilament + and synaptophysin + phagocytosed material and were in close proximity to C3b+ stressed/damaged cortical neurons (Figure 3; Watkins et al., 2016), suggesting that some of these cells contribute to worsening tissue damage.

C5aR1 activation, via the induced production of interferon γ and TNFα by macrophages and other monocytes (Pandey et al., 2017; Serfecz et al., 2021), may be one mechanism by which activated complement elicits damage. The induced expression of such cytokines can be blocked with the brain penetrant C5aR1 inhibitor PMX53 (Serfecz et al., 2021). Other small molecule antagonists of C5aR1 are effective in abrogating neuroinflammation, inflammasome activation and neurodegeneration in vivo, are brain penetrant and non-toxic. Eculizumab, a monoclonal antibody approved for use in paroxysmal nocturnal hemoglobinuria, prevents C5 convertase cleaving C5 into C5a. Trials of eculizumab in neuromyelitis optica have been encouraging (Pittock et al., 2019). Avdoralimab, a monoclonal antibody designed to block C5a binding to C5aR1, can prevent neutrophil migration into lungs and blocked inflammatory cytokine release in COVID-19 infection (Woodruff and Shukla, 2020). Therefore, these distinct pharmaceutical approaches to reduce C5aR1 activation may represent useful mechanisms to modulate resident microglia and other C5aR1 expressing cells in MS (Vergunst et al., 2007; Michailidou et al., 2018; Carpanini et al., 2019; Schartz and Tenner, 2020).

Cultured astrocytes and microglia can be induced to synthesise complement components, including C1q, C3, and factor B, when exposed to CSF from lymphotoxin-α treated rats. Lymphotoxin-α is elevated in concentration in the MS CSF and when over-expressed in the leptomeninges, recapitulates the pattern of subpial demyelination seen in disease (James Bates et al., 2022). Therefore, complement activation in the connective tissue spaces and parenchyma can stimulate the production of TNFα and other pleiotropic cytokines, which can be directly cytotoxic (and differentially expressed in cases characterised by leptomeningeal inflammation and more extensive cortical demyelination) (Magliozzi et al., 2019b; Picon et al., 2021), whilst cytokine release by activated glia can further drive the transcription of genes encoding early complement proteins. Future work, for example, could assess the effect of blocking C5aR1 activation in models of leptomeningeal inflammation and subpial cortical demyelination.

Microglia can induce a reactive astrocyte response in vitro, in part requiring IL-1β, TNFα and C1q, that is associated with a neuroinflammatory and non-homeostatic response and the elevated transcription of C3 and other signature genes (Liddelow et al., 2017). C3+ astrocytes are seen in actively demyelinating MS WM lesions and C3b+ astrocytes, some with a dysmorphic appearance, were frequently observed in subpial and peri-vascular sites in our MS GM (Supplementary Figure 2). The expression of C3 is not by itself an indicator of the neurotoxic potential of an astrocyte and such findings should be treated cautiously (Escartin et al., 2021). Indeed, we found the density of C3b+ astrocytes were unchanged between control and MS, whilst conversely, the density of C3b-/GFAP+ astrocytes were significantly reduced at subpial and perivascular GM lesions. These data demonstrate alterations in astrocytes occur in association with complement activation at or near the cortical GM.

Our current study is limited by the non-availability of neuroinflammatory disease control tissues harbouring frank inflammatory lesions in the selected blocks of interest, which were chosen as they anatomically matched the available MS blocks. A further limitation was that the assessment of complement immunostaining was performed semi-quantitatively and was not automated. The presence of staining artefacts of the delicate leptomeninges could be recognised by the investigator but was challenging to quantify digitally.

The presence of elevated levels of complement C1q, C3b, C4d, and Bb, and the release of anaphylatoxins that engage C3aR1 and C5aR1 expressed by microglia, may stimulate functional pathways leading to the induced expression of inflammatory cytokines and phagocytic processes in the MS grey matter. Therefore, complement activation of anaphylatoxin receptor bearing microglia/macrophages represents a modifiable pathway to abrogate the extent of compartmentalised inflammation and cortical grey matter pathology to improve the outcome for some people with progressive MS.

The original contributions presented in this study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the South Wales Research Ethics Committee (study numbers 08/MRE09/31+5 and 13/WA/0292). The patients/participants provided their written informed consent to participate in this study.

RE, LW, BM, JN, and OH: methodology, investigation, writing – original draft, review and editing, and funding. KH, GS, CD, MN, and MD: methodology, investigation, and writing – review and editing. MR and DF: resources and writing – review and editing. All authors contributed to the article and approved the submitted version.