Most people have likely experienced the discomfort of an eyelash falling onto the surface of their eye. Or that gritty sensation when dust blows into the eye and irritates the surface. These sensations are warnings from sensory nerves in the cornea, the transparent tissue that covers the iris and pupil. Corneal nerves help regulate blinking, and control production of the tear fluid that protects and lubricates the eye.

But if the cornea suffers damage or infection, it can become inflamed. Long-lasting inflammation can damage the corneal nerves, leading to pain and vision loss. If scientists can identify how this happens, they may ultimately be able to prevent it. To this end, Royer et al. have used mice to study three causes of hard-to-treat corneal inflammation. The first is infection with herpes simplex virus (HSV-1), which also causes cold sores. The second is eye allergy, where the immune system overreacts to substances like pollen or pet dander. And the third is graft-versus-host disease (GVHD), an immune disorder that can affect people who receive a bone marrow transplant.

Royer et al. showed that HSV-1 infection and GVHD – but not allergies – made the mouse cornea less sensitive to touch. Consistent with this, microscopy revealed damage to corneal nerves in the mice with HSV-1 infection and those with GVHD. Further experiments showed that immune cells called CD4 T cells and a protein called complement C3 were contributing to this nerve damage. Treating the mice with an experimental drug derived from cobra venom protected the cornea from the harmful effects of inflammation. It did so by blocking activation of complement C3 at the eye surface.

Identifying factors such as complement C3 that are responsible for corneal nerve damage is an important first step in helping patients with inflammatory eye diseases. Many drugs that target the complement pathway are currently under development. Some of these drugs could potentially be adapted for delivery as eye drops. But first, experiments must test whether complement also contributes to corneal nerve damage in humans. If it does, work can then begin on testing these drugs for safety and efficacy in patients.

Dysregulated complement activation is increasingly recognized as a significant pathological event in a variety of neurodegenerative and neuroinflammatory diseases of the central nervous system (Tenner et al., 2018). These include conditions from Alzheimer’s disease and age-related macular degeneration (AMD) to amyotrophic lateral sclerosis and multiple sclerosis (Hong et al., 2016; Knickelbein et al., 2015; Lee et al., 2018; Loveless et al., 2018). Complement may orchestrate peripheral nervous system (PNS) disorders as well. For instance, nociceptive hypersensitivities, sensory neuropathies, and Guillain-Barré syndrome have been linked to aberrant complement activation (Rosoklija et al., 2000; Ramaglia et al., 2008; Jang et al., 2010; Fritzinger and Benjamin, 2016; Xu et al., 2018; Susuki et al., 2007). However, the mechanistic role of complement is not well understood in neuroinflammatory pathologies impacting the PNS. Moreover, the pathophysiological trigger of sensory fiber retraction which contributes to sensation loss and in some instances chronic pain is unclear (Stepp et al., 2017). Available treatments for peripheral neuropathies primarily focus on clinical symptoms without addressing the underlying pathomechanisms (Colloca et al., 2017). Accordingly, elucidation of relevant pathomechanisms underlying sensory neuropathies may translate into efficacious mechanism-based therapeutics.

The complement cascade is comprised of dozens of soluble and membrane-bound factors essential for host defense and efficient clearance of cellular debris. However, proper regulation of complement activation is necessary to balance immune responses and prevent collateral tissue damage (Ricklin et al., 2016). The complement cascade pivots upon activation of complement component 3 (C3) for its downstream proinflammatory effects. The pathogenic contributions of complement in systemic disease have been reviewed extensively in recent years (Dobó et al., 2018; Hajishengallis et al., 2017; McGeer et al., 2017; Ricklin et al., 2016). Nonetheless, emerging concepts centering on complement effector synthesis within inflamed tissue microenvironments have ushered in a ‘renaissance’ of novel complement-targeted drug development strategies (Ricklin et al., 2018; Tomlinson and Thurman, 2018). Therefore, elucidating whether the complement cascade is a viable therapeutic target in inflammation-associated peripheral nerve impairment remains medically important.

The eye has been labeled a ‘complement dysregulation hotspot’ due to complement’s contributions to many ophthalmic diseases, but complement activation is restricted in the healthy cornea (Clark and Bishop, 2018). As observed in AMD, subtle inflammatory reactions in the eye can mediate significant visual morbidity. Accordingly, ocular inflammation is tightly regulated by various anatomic, physiologic, and immunologic mechanisms in order to maintain visual acuity (Amouzegar et al., 2016; Streilein, 2003; Taylor et al., 2018). These homeostatic mechanisms, collectively dubbed ‘ocular immune privilege,’ help preserve the ocular surface and enable the cornea to properly focus incoming light onto the retina. Furthermore, corneal nerves are increasingly recognized as central regulators of immune privilege at the ocular surface (Guzmán et al., 2018; Neelam et al., 2018; Paunicka et al., 2015). Consequently, inflammatory events that damage corneal nerves can have insidious consequences in terms of ocular surface health and transparency (Müller et al., 2003; Shaheen et al., 2014; Stepp et al., 2017). One such pathway that can rapidly initiate inflammation following activation is the complement cascade. Corneal nerves originate predominantly from the ophthalmic branch of the trigeminal ganglion (TG), and these peripheral nerves provide the cornea with the highest density of sensory fibers in the human body. While the cornea and peripheral nerves constitutively synthesize complement proteins (Bora et al., 2008; de Jonge et al., 2004), the role of the complement cascade in corneal nerve damage has not been explored. Nonetheless, the complement pathway is poised for vigorous activation in the cornea in response to noxious or inflammatory stimuli (Bora et al., 2008). Moreover, C3 activation has been reported in the cornea soon after infection with herpes simplex virus type 1 (HSV-1), which is a common cause of corneal sensory nerve damage in patients (Royer et al., 2017; Sacchetti and Lambiase, 2014). Given the cornea’s high density of sensory nerves, it is particularly amenable for investigating the mechanistic role of complement in peripheral nerve damage. To this end, we evaluated corneal nerve integrity and mechano-sensory function using a murine model of ocular HSV-1 infection to test the hypothesis that local complement activation and T cell engagement coordinate corneal nerve damage.

The pathobiology underlying non-penetrating corneal nerve damage is not well understood, although inflammation is generally recognized as an important feature (Neelam et al., 2018; Shaheen et al., 2014; Cruzat et al., 2015; Chucair-Elliott et al., 2016; Chucair-Elliott et al., 2017b; Chucair-Elliott et al., 2017a). While HSV-1 is a clinically prominent cause of corneal nerve damage and sensation loss, a variety of pathogenic microbes impair the corneal nerve architecture upon ocular infection (Cruzat et al., 2015). This observation adds to evidence that the neurotropism of HSV-1 is not directly responsible for nerve damage (Chucair-Elliott et al., 2016). In addition to sensation loss evoked by HSV-1, corneal nerve alterations in other contexts such as dry eye disease are associated with a broad array of neuropathic clinical symptoms including dryness, itch, and pain (Andersen et al., 2017). To further qualify the possible role of complement in corneal nerve damage independent of infection, we evaluated corneal mechanosensory function in two noninfectious T cell-dependent ocular surface inflammatory diseases. For this purpose, we utilized established murine models of allergic eye disease (AED) and ocular graft-versus-host disease (GVHD) (Herretes et al., 2015; Lee et al., 2015). Our rationale for this is that complement has been implicated in the etiology of systemic GVHD and allergic inflammation (Gour et al., 2018; Kwan et al., 2012; Ma et al., 2014; Nguyen et al., 2018; Zhang and Köhl, 2010). However, a neuroinflammatory role of complement has not been described for either disease within the eye.

The translational relevance of this study is underscored by in vivo confocal microscopy data from multiple clinical studies showing architectural changes in the corneal nerves of patients with herpetic keratitis, chronic ocular allergy, and ocular GVHD (Hamrah et al., 2010; Müller et al., 2015; Moein et al., 2018; Hu et al., 2008; Le et al., 2011; Leonardi et al., 2012; Tepelus et al., 2017; He et al., 2017a). Elucidating the pathomechanisms underlying corneal nerve damage may enable development of more effective therapeutics to mitigate progression of such ocular surface inflammatory diseases. Each of the animal models utilized herein mimic clinically important, chronic ocular surface morbidities. Herpes simplex virus type 1 (HSV-1) is a common cause of neurotropic keratitis and remains a leading cause of infectious corneal blindness (Sacchetti and Lambiase, 2014; Farooq and Shukla, 2012). The incidence of ocular allergy exceeds twenty percent of the population with varying degrees of neurogenic ocular surface discomfort that can severely diminish quality of life (Craig et al., 2017; Patel et al., 2017; Saban et al., 2013). Finally, GVHD is the greatest cause of non-relapse morbidity following hematopoietic stem cell transplantation (HSCT) used to treat life-threatening malignancies and immunologic diseases (MacDonald et al., 2017). A majority of patients with chronic presentations of GVHD suffer from ocular surface involvement (Shikari et al., 2013).

Mechanistic advances in our understanding of the complement cascade’s physiologic regulation and pathologic contributions in disease have predictably spawned substantial investments in complement-targeted drug development over the past decade (Harris et al., 2018; Ricklin et al., 2018; Tomlinson and Thurman, 2018). The ocular surface is a unique milieu in which pharmacologic modulation of the complement pathway may limit the severity of inflammatory disease and improve clinical outcomes. This study provides proof of concept that such interventions may have important clinical impacts on ocular surface disease, and specifically neuropathic sensory disorders affecting the cornea. Complement-targeted drug development for ophthalmic use has almost exclusively focused on AMD with some interest in neuromyelitis optica (NMO), Stargardt disease, and autoimmune uveitis (Harris et al., 2018; Pittock et al., 2013). By July 2019, there were no complement-specific therapeutics indicated for ocular surface use registered on www.clinicaltrials.gov (search strings: ‘complement’ / ‘C3’ / ‘C5’ / ‘eculizumab’ AND ‘cornea’, ‘conjunctiva’, ‘dry eye’, ‘keratitis’, OR ‘ocular surface’). Nonetheless, the complement pathway is implicated in the pathophysiology of several ocular surface diseases (Bora et al., 2008). The current investigation provides an important advancement in this arena by demonstrating that dysregulated complement activation can specifically contribute to sensory nerve damage in the cornea.

While corneal nerves are increasingly implicated in maintenance of corneal immune privilege (Paunicka et al., 2015; Neelam et al., 2018; Guzmán et al., 2018), this study is the first to identify that C3 is involved in the pathobiology of corneal sensory nerve damage. However, the individual fates of damaged corneal nerves were not explored herein. Lessons from peripheral nerve injury in other settings indicate that some damaged nerves undergo apoptosis, yet effectively promoting functional regeneration of surviving neurons remains a major clinical hurtle (Doron-Mandel et al., 2015; Menorca et al., 2013; Scheib and Höke, 2013; Shacham-Silverberg et al., 2018). Likewise, promoting functional regeneration of damaged corneal nerves is important for restoration of immune privilege and ocular surface health. Our approach evaluated sensory nerve damage in murine models of T cell-dependent ocular surface disease including HSV-1 keratitis, OVA-induced AED, and ocular GHVD. The lack of a sensory phenotype in the AED model was unanticipated in light of evidence that patients with severe allergy exhibit changes in corneal nerve morphology (Hu et al., 2008; Le et al., 2011; Leonardi et al., 2012). This contrasting model underscores the context-dependency of sensation loss during corneal inflammation.

While we focused on C3 in HSV-1 keratitis and ocular GVHD due to the direct link to overt corneal sensation loss, complement may still have an important role in mediating neurogenic hypersensitivities in AED. In addition to loss of mechano-sensory function, corneal nerve pathologies can involve a broad array of neuropathic clinical symptoms including dryness, itch, and pain (Andersen et al., 2017). Inflammatory mediators facilitate neuropathic hypersensitivities, but the therapeutic potential of complement inhibition is often overlooked (Baral et al., 2019; Fritzinger and Benjamin, 2016). Moreover, the cornea produces many neurotrophic factors that influence sensory innervation in health and disease (Sacchetti and Lambiase, 2017; Yu et al., 2015). Given the involvement of C3 activation in sensory nerve damage, future investigation into crosstalk between complement and other neurotropic factors in the cornea is warranted. Furthermore, AED is driven primarily by a combined Th2/Th17 CD4 T cell response that differs from the Th1 bias observed in HSV-1 keratitis and ocular GVHD (Herretes et al., 2015; Saban et al., 2013; Tang and Hendricks, 1996). This suggests that Th1 cytokines such as IFNγ, IL-2, and lymphotoxin-alpha may be important in coordinating corneal sensation loss. Nonetheless, the unique inflammatory milieus and differential polarizations of cornea-infiltrating T cells across these disease models may also influence the downstream effects of complement activation in the tissue microenvironment (Tomlinson and Thurman, 2018). Future studies are needed to broaden the scope of these observations to other peripheral nerve diseases. For example, complement-mediated microvascular damage in diabetes correlates with neuropathy (Rasmussen et al., 2018; Rosoklija et al., 2000). While diabetic peripheral neuropathy can lead to neurotropic corneal ulcerations (Gao et al., 2016b), the role of complement in diabetic corneal disease is currently unknown.

Complement enhances clearance of infected cells and nascent virions during HSV-1 infection (Kostavasili et al., 1997; Lubinski et al., 2002; McNearney et al., 1987). Importantly, HSV-1 can also evade C3 activation through endogenous expression of glycoprotein C (gC). By limiting C3b deposition on infected cells and nascent virions, gC can inhibit subsequent C5 binding and membrane attack complex (MAC) formation (Kostavasili et al., 1997; Lubinski et al., 2002; McNearney et al., 1987; Tegla et al., 2011). This could explain why herpetic keratitis is less severe in rabbits infected with a gC-deficient strain of HSV-1 during acute infection (Drolet et al., 2004). However, this gC-mediated viral evasion strategy may also be cell type-dependent. Cell culture modeling indicates that neuronal cells maintain host-encoded complement regulator expression more efficiently and are more resistant to MAC deposition than epithelial cells upon HSV-1 infection (Rautemaa et al., 2002). Accordingly, complement-mediated sensory nerve damage in HSV-1 keratitis may reflect a maladaptive outcome of host-defense. This is consistent with our data showing that immunologically naive C3^-/- mice maintain corneal sensation and innervation, yet they exhibit concomitant enhancement of viral shedding in the tear film and increased viral burden in the TG by day 7 p.i. The absence of a concordant difference in HSV-1 titers in corneas from WT and C3^-/- mice is likely explained by the potent antiviral effects of type-1 interferon (Conrady et al., 2011; Royer and Carr, 2016). This suggests that the maintenance of corneal innervation in C3^-/- mice enables enhanced shedding of nascent virions produced within the TG. Moreover, there is no difference in the amount of latent HSV-1 in the TG of naive WT and C3^-/- mice 30 days after ocular HSV-1 challenge (Royer et al., 2019). Taken together, this evidence indicates that although C3 is involved in progression of keratitis and sensory nerve fiber retraction during acute infection, it likely does not contribute to complete elimination of infected nerves.

The studies reported herein involve immunologically naive mice, but the impacts of preexisting humoral immune responses on complement pathway activation and regulation in the cornea during HSV-1 infection remain incompletely understood. This point is of considerable importance clinically, as herpes-associated neurotropic keratitis typically develops as a result of recurrent corneal infections in patients (Hamrah et al., 2010). Furthermore, we have recently reported that C3 is essential for optimal antibody-dependent viral clearance following ocular HSV-1 challenge in mice (Royer et al., 2019; Royer et al., 2017). While vaccinated animals did not exhibit corneal sensation loss following ocular HSV-1 infection in those studies, deposition of the terminal C3 cleavage product C3d was present in the corneal epithelium (Royer et al., 2017). Host gene expression data herein corroborates that HSV-1 infection creates an imbalance in the local complement effector to regulator expression ratios that contribute to aberrant complement pathway activation in the cornea. This imbalance in ‘complement proteostasis’ may favor deposition of complement fragments and sublytic MAC on corneal sensory nerve fibers (Tegla et al., 2011; Triantafilou et al., 2013). Notwithstanding, the nerve-intrinsic molecular mechanisms responsible for trigeminal sensory fiber retraction, neuronal death, or axonal regeneration in the cornea are largely unknown (Stepp et al., 2017). Future work is needed to identify the respective complement activation pathways involved in pathologic and protective immune responses to HSV-1 in the cornea.

Identification of the complement pathway’s role in initiating corneal sensation loss following HSV-1 infection is an important advancement in the mechanistic understanding of this disease process, as it introduces an array of potential drug targets for local therapy. Previous observations note that complement activation in antigen-induced keratitis may be mediated by cellular immune mechanisms (Verhagen et al., 1992). The identification of CSF1R⁺ macrophages/monocytes as a local source of C3 during infection (Figure 4D) is also critical, as these cells are recruited to the cornea in the early stages of HSV-1 infection and are associated with corneal nerve damage in HSV-1 keratitis (Chucair-Elliott et al., 2017b; Conrady et al., 2013). Moreover, CD4 T cells coordinate this pathology. Our data corroborate findings from the Hendricks’ lab showing that CD4 T cells are associated with corneal sensation loss in HSV-1 keratitis (Yun et al., 2014), yet our adoptive transfer experiments establish that this process is not dependent upon endogenous C3 production by cornea-infiltrating donor T cells (Figure 2D). Notably, the tempo of HSV-associated sensation loss was consistent among models once initiated (Figure 1B, Figure 2D, Figure 3A). However, the initial onset was delayed by one day in the adoptive transfer model. This likely reflects the relative lymphopenic status of T cell-reconstituted TCRα^-/- mice compared to WT mice (compare Figure 2B and Figure 3—figure supplement 1).

Corneal sensation loss in HSV-1 infection and ocular GVHD shared a common mechanism dependent upon CD4⁺ T cells and complement C3. The role of intracellular C3 in regulating human Th1 responses has received much attention in recent years (Elvington et al., 2017; Hansen et al., 2019; Liszewski et al., 2013). Such studies involve CD3 and CD46 stimulation to activate human CD4 T cells in vitro. However, mice do not express CD46. Instead, anaphylatoxin receptor signaling has been shown to modulate Th1 cytokine production in murine T cells (Strainic et al., 2008). Our ex vivo re-stimulation data clearly show that there is no defect in IFNγ production by CD4 T cells from C3^-/- mice following HSV-1 infection. Others have shown similar data based on T cell proliferation in response to re-stimulation with HSV-1 antigen (Da Costa et al., 1999). In contrast, a reduction in IFNγ production by CD4 T cells from C3^-/- mice was reported following in vitro expansion under Th1 polarizing conditions (Liszewski et al., 2013). Nonetheless, it is possible that anaphylatoxin receptor signaling from locally produced C3a (and potentially C5a generated downstream) modulates T cell effector function upon extravasation into the inflamed cornea. The precise relationships between T cells and C3 in our mouse models of corneal neuropathic disease remain to be identified. Our working hypothesis involves an indirect mechanism whereby sublytic MAC deposition from local complement activation damages sensory nerves, which are then cleared by T cell-activated phagocytes or other cytolytic cells. These could include tissue-resident and infiltrating leukocytes (macrophages, monocytes, dendritic cells, and NK cells) as well as nerve-associated corneal epithelial cells (Buela and Hendricks, 2015; Chucair-Elliott et al., 2017b; Gao et al., 2016a; Koyama and Hill, 2016; Liu et al., 2017; Royer et al., 2018; Seyed-Razavi et al., 2014; Stepp et al., 2017). Emerging evidence indicates that the plasma membranes of corneal nerves and epithelial cells fuse thereby enabling cytosolic exchange (Stepp et al., 2017). In light of this, it is plausible that complement-mediated damage to corneal epithelial cells has a direct impact on sensory nerves.

Although corneal nerve damage is an established pathology in animal models of HSV-1 infection (Chucair-Elliott et al., 2015; He et al., 2017b; Yun et al., 2014), this is the first report documenting loss of corneal mechano-sensory function in ocular GVHD. Notably, patients rarely exhibit neurological manifestations of GVHD affecting other tissues/organs (Grauer et al., 2010). Corneal sensation loss may provide a clinical benchmark for initiation of targeted therapies to curb progression of ocular GVHD in patients. Data from our animal model of GVHD suggest that corneal sensation loss is an early warning sign of progressive ocular and systemic GVHD. Corneal sensation measurements are warranted in patients following HSCT to substantiate this finding among those who develop chronic GVHD. Published imaging studies confirm that corneal nerve remodeling occurs in patients with ocular GVHD (He et al., 2017a; Tepelus et al., 2017); therefore, clinical studies to delineate the kinetics of disease onset are needed. Complement C3 is a known driver of systemic GVHD (Kwan et al., 2012; Ma et al., 2014; Seignez et al., 2017), and our data show that aberrant complement pathway activation also contributes to the pathogenesis of ocular GVHD including corneal sensation loss. Furthermore, localized CVF treatment preserved corneal sensation in both sexes during ocular GVHD, but treatment only abrogated other facets of ocular surface disease in female mice. This dimorphic outcome may reflect insufficient maintenance of C3 depletion in the ocular surface microenvironment, as animals were only treated twice weekly. Furthermore, complement activity is reportedly limited by terminal pathway components in female mice compared to males (Kotimaa et al., 2016). Collectively, our data suggest that sensory nerve involvement may be a unique facet and treatment target in ocular GVHD.

While the complement pathway has many components, C3 is the heart of the pathway and its activation products mediate virtually all downstream pathway functions. Accordingly, we used CVF to target complement activation in models of HSV-1 keratitis and ocular GVHD as a proof of concept for local delivery of complement-targeted therapeutics for ocular surface disease. In contrast to the neurotoxic effects of complete cobra venom, purified CVF is a ‘nontoxic’ derivative. CVF forms a biochemically stable convertase to rapidly hydrolyze mammalian C3 and C5. This ultimately results in complement inactivation via effector consumption (Vogel and Fritzinger, 2010). The only recorded side effect of purified CVF administration in rodents is transitory anaphylatoxin-mediated pulmonary inflammation resulting in acute respiratory distress (Proctor et al., 2006; Vogel and Fritzinger, 2010). In our hands, respiratory distress was observed in some mice following the initial sub-conjunctival CVF injection irrespective of group assignment (animals were euthanized). However, topical CVF administration did not provoke respiratory or corneal abnormalities in any animals. Previous findings also show that topical CVF administration has no obvious effects on corneal health or transparency (Zaidi et al., 2010).

Experimental CVF administration was once thought to directly stimulate T cells, but such effects were later attributed to mitogen contamination (Rumjanek et al., 1978; Cauvi et al., 2012). Because our disease models were T cell-dependent, we utilized highly-purified CVF from a commercial source for our studies. Moreover, we found no evidence to suggest that CVF impacted T cell responses outside of the eye. Local CVF treatment reduced corneal T cell infiltration following HSV-1 infection (Figure 5G). However, this is likely due to reduced inflammation overall in C3-depleted corneas (Figure 5D). Likewise, ocular CVF treatment in the GVHD model did not impact on the total numbers of T cells in the corneas at the experiment endpoint. However, we have not excluded the possibility that CVF treatment may delay the tempo of T cell influx. Nonetheless, CVF administration preserved corneal sensation in both disease models. By addressing the ‘heart’ of the complement pathway (C3), this study opens the door to future investigation to further elucidate the relevant pathomechanisms and corresponding therapeutic targets. The relevant complement activation pathways (classical, alternative, lectin, terminal), cellular targets, and impacts of anaphylatoxin receptor signaling are topics of future investigation.

Complement activation is a double-edged sword in the cornea, as activation can be either protective or harmful. Low-level complement turnover is observed in healthy corneas/tears, and complement may even contribute to the relative ‘paucibacterial’ state of the ocular surface microbiome (Doan et al., 2016; McDermott, 2013; Willcox et al., 1997). The mechanisms regulating each complement-mediated effect remain incompletely understood. Elucidation of how complement activation is triggered, the relevant molecular mechanisms of its downstream signaling, and contributions to pathology will promote an important advancement in drug development for ocular surface disease. Animal models are necessary for mechanistic studies when it comes to understanding complement pathway regulation, especially in dynamic tissue microenvironments; however, differences in complement regulation exist between mouse and man that will also have to be resolved to further assess the translational potential (Jacobson and Weis, 2008). This certainly applies to putative complement-targeted therapeutics for ocular surface treatment. Even among individual patients, small nucleotide polymorphisms evoke changes in complement activity that have major impacts on disease risks. Such genetic linkages, dubbed ‘complotype,’ are important in differential susceptibility to AMD (Harris et al., 2012; Paun et al., 2016). Accordingly, complotype differences may help explain differential susceptibility patterns in a variety of ocular surface diseases. Our data herein have demonstrated successful prophylactic intervention in repressing complement-mediated sensory nerve pathology in two well characterized experimental T cell-dependent corneal disease models. Other avenues of future investigation are also needed to determine the efficacy of therapeutic intervention on established ocular surface diseases. Successful implementation of complement-targeted therapeutics for topical ophthalmic use may provide the benefit of controlling insidious ocular surface diseases without the risks associated with systemic therapies.

Data generated during this study are included in the manuscript.

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Thank you for submitting your article "Complement and CD4⁺ T cells drive context-specific corneal sensory neuropathy" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Tadatsugu Taniguchi as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Mihaela Gadjeva (Reviewer #2); Susmit Suvas (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

In this interesting study, Derek et al. showed the involvement of complement C3 in causing corneal sensory neuropathy in mouse models of HSV keratitis, T-dependent allergic eye disease, and ocular graft-versus-host disease (GVHD). They find that complement C3 plays a key role in nerve damage during HSV-1 induced ocular keratitis, as well as in a mouse model of GVHD. They also find that T cells played a role in both disease conditions, and in particular CD4 T cells. Local depletion of C3 by cobra venom factor or the use of C3^-/- mice in these two models of chronic ocular inflammation prevented the loss of corneal sensation. The authors also showed that adoptive transfer of purified splenic CD4⁺ or CD8⁺ T cells from HSV-1 infected mice into TCRa^-/- mice documented the involvement of CD4 but not CD8 expressing T cells in causing the loss of corneal sensation after ocular HSV-1 infection. Together, these studies confirm the involvement of C3 and CD4 expressing T cells in causing the loss of corneal sensation in two major mouse models of chronic ocular inflammation. Therefore, these findings uncover immune mechanisms of nerve damage in several important eye inflammation contexts, and has interesting clinical implications for major conditions for patients that suffer from HSV1 and GVHD. All three reviewers agreed that this is a nice study and worthy of publication. However, there is a need to address some points and concerns raised below.

Essential revisions:

1) What is the relationship between C3 and T cells in HSV1 induced neuropathy? Figure 1F does not convincingly show equal (or decreased) influx of CD3+ T cells in HSV1 infected corneas from B6 and C3^-/- mice. There is a need to prove this by a more quantitative approach either by imaging or by flow cytometry.

2) The authors did not address potential non-specific effects of CVF while administering into infected corneas. Does it affect T cell survival or influx in HSV infected corneas?

3) Does CVF treatment decrease local T cell recruitment in the GVHD model? Figure 8E seems to indicate this, but can the authors quantify this by immunostaining or flow cytometry?

4) The baseline role for T cells in mediating HSV1 induced pain and neuropathy is not complete prior to adoptive transfers. In the HSV1 model, how do WT mice compare with TCRa^-/- mice for corneal sensation loss in the Cochet-Bonnet score? Is there less nerve damage in TCRa^-/- mice?

5) In Figure 3B, can the authors show the full time course of the corneal sensation threshold between WT vs. OT-II transfer data?

6) Generally, is the decrease in neuronal fibers associated with loss of neuronal cells in the TGs?

7) In view of recent work describing the importance of intracellular complement for T cell functionality, authors should interrogate the importance of intracellular complement synthesis and/or T cell activation in the models (Hansen et al., 2019; West et al., 2018). Indeed, authors show RT-PCR data from day 3 sorted cells from mice infected with HSV corneas, however, the dominant T-cell mediated effect seems to occur at later time points, suggestive a possible contribution of endogenous T-cell derived complement. Can the authors address this possibility?

8) If C3 is predominant factor in causing the loss of corneal sensation in these models, what is the possible underlying mechanism?

9) Can authors comment on the potential mechanisms of neuronal fiber retractions?

10) The use of CVF treatment offers a traditional approach to reduce complement activation. Given the developed therapies that target C5aR-signaling, authors should comment on the use of anti-C5a biologics for complement blockade in view of their discussion on potential therapies.

11) What possible factors from effector CD4 T cells are involved in activating C3 in GVHD and keratitis model?

12) While AED seems to be a contrasting model, the authors find lack of sensory phenotypes and nerve loss in this model (Figure 6). Therefore, we do not think this is a good model as comparison. The limitations of comparing this vs. the other models should be Discussed.

13) In Figure 1, it's interesting that while HSV-1 titers go up in the tear film and trigeminal ganglia (TG) at days 5 and 7 in C3^-/- mice compared to WT mice, HSV titers are not elevated in the C3^-/- cornea compared to WT mice. It's possible that the reason viral burdens are similar between WT and c3^-/- cornea is that a large number of nerve fibers and keratinocytes are destroyed that harbor the virus in WT mice. Could the authors address this point in the Discussion?

14) References: The authors can cite additional papers relevant to HSV-1 and complement C3: This includes the papers by Carroll and Knipe showing myeloid immune cell derived complement plays a role in HSV-1 host defense in peripheral tissues (Verschoor et al., 2001; Gadjeva et al., J Immunol. 2002 PMID: 12421924; Verschoor et al., J Immunol 2003 PMID:14607939) and papers showing that HSV-1 inhibits complement through glycoprotein C to evade host defense (Hook et al., J Virol 2006 PMID: 16571820; Lubinski et al., 2002)

Essential revisions:

1) What is the relationship between C3 and T cells in HSV1 induced neuropathy? Figure 1F does not convincingly show equal (or decreased) influx of CD3+ T cells in HSV1 infected corneas from B6 and C3^-/- mice. There is a need to prove this by a more quantitative approach either by imaging or by flow cytometry.

We performed additional experiments to quantitatively assess T cell in the corneas of WT and C3^-/- mice by flow cytometry. These data show no difference in CD4 or CD8 T cell infiltration at day 7 post-infection(Figure 1G).

Briefly, the relationship between C3 and T cells is likely an indirect, coordinated process. Please see our working hypothesis for the underlying pathomechanism in critique #8 below.

2) The authors did not address potential non-specific effects of CVF while administering into infected corneas. Does it affect T cell survival or influx in HSV infected corneas?

Pharmacologic information about CVF is now included in the Discussion to clarify the mechanism of action and potential side effects. Briefly, CVF is generally nontoxic. While it has been used experimentally for several decades, there is little substantive evidence that it directly impacts the physiology of T cells. Please see Discussion paragraphs nine and ten for more information.

CVF treatment leads to a ~50% reduction in cornea-infiltrating CD4 T cells compared to the vehicle control group (Figure 5G). This is consistent with the observation that CVF treatment also reduces corneal inflammation/edema (Figure 5D).

CVF treatment had no discernable impact on T cell expansion in the draining lymph nodes or numbers in peripheral circulation (Figure 5—figure supplement 2). Taken together, this suggests that CVF does not alter T cell survival.

3) Does CVF treatment decrease local T cell recruitment in the GVHD model? Figure 8E seems to indicate this, but can the authors quantify this by immunostaining or flow cytometry?

T cell numbers and nerve densities were quantified from confocal images of cornea flat mounts using Imaris software (AED and GVHD models). These data are now included in Figures 6, 7, and 8.

Briefly, no differences were found in the numbers of CD3⁺ cells in the central corneas of CVF vs. PBS treated mice in the GHVD model at the experiment endpoint (Figure 8H). Given the protracted treatment regimen, this suggests that CVF likely has no impact on T cell survival (relevant to critique # 2 above).

Our data does not exclude the possibility that CVF treatment may delay T cell influx. However, the Perez laboratory is currently investigating T cell response dynamics in the progression of ocular GVHD.

4) The baseline role for T cells in mediating HSV1 induced pain and neuropathy is not complete prior to adoptive transfers. In the HSV1 model, how do WT mice compare with TCRa^-/- mice for corneal sensation loss in the Cochet-Bonnet score? Is there less nerve damage in TCRa^-/- mice?

Cochet-Bonnet scores for corneal sensation are highly reproducible direct measurements (data are not relative/normalized). As shown in the original manuscript, sensation loss was not observed in TCRα^-/- mice without addition of exogenous T cells.

Corneal nerve imaging was not performed on corneas from TCRα^-/- mice, but the sensation data underscore that HSV-associated sensation loss is T cell-dependent.

The initial time (day post-infection) at which sensation is ‘significantly’ reduced relative to the corresponding control lags by one day in the adoptive transfer model compared to WT mice. This corresponds with the relative lymphopenic status of the TCRα^-/- mice following adoptive transfer compared to WT mice, which have more than 10 times as many T cells in the cornea-draining lymph nodes at day 8 post-infection (compare Figure 2B and Figure 2—figure supplement 1A). However, the tempo of sensation loss is consistent between models once instigated by HSV-1 infection.

From a gross pathology standpoint, corneas from TCRα^-/- control mice generally remained transparent following HSV-1 infection. However, corneal opacification was observed in HSV-1-infected TCRα^-/- mice reconstituted with exogenous T cells (data not shown). These observations parallel those from the Hendricks lab showing a reduction in corneal opacification when mice are depleted of CD4 T cells (Yun et al., 2014).

5) In Figure 3B, can the authors show the full time course of the corneal sensation threshold between WT vs. OT-II transfer data?

Baseline sensation and control data are now included in Figure 3B and Figure 3—figure supplement 2.

These experiments were performed to determine the necessity of antigen-specific T cells in driving sensation loss after the tempo of corneal sensation loss had been established (Figure 1B and Figure 3A). Accordingly, corneal sensation data were only measured at baseline and day 7 post-infection for the experiments involving OT-II mice and cells.

6) Generally, is the decrease in neuronal fibers associated with loss of neuronal cells in the TGs?

The cell bodies of the corneal nerves are housed within the trigeminal ganglion. Accordingly, addressing these related issues would require meticulous nerve tracing studies well beyond the scope of the current manuscript.

The current investigation specifically addresses immune-mediated mechanisms of corneal sensation loss using murine models of clinically-relevant ocular surface inflammatory diseases. While certainly important, the individual fates of damaged corneal sensory nerves, were not explored.

The revised Discussion includes a brief synopsis of the relevant literature:

“…the individual fates of damaged corneal nerves were not explored herein. Lessons from peripheral nerve injury in other settings indicate that some damaged nerves undergo apoptosis, yet effectively promoting functional regeneration of surviving neurons remains a major clinical hurtle… Likewise, promoting functional regeneration of damaged corneal nerves is important for restoration of immune privilege and ocular surface health.”

7) In view of recent work describing the importance of intracellular complement for T cell functionality, authors should interrogate the importance of intracellular complement synthesis and/or T cell activation in the models (Hansen et al., 2019; West et al., 2018). Indeed, authors show RT-PCR data from day 3 sorted cells from mice infected with HSV corneas, however, the dominant T-cell mediated effect seems to occur at later time points, suggestive a possible contribution of endogenous T-cell derived complement. Can the authors address this possibility?

Intracellular complement function is intriguing and certainly noteworthy. However, this area remains somewhat controversial within the complement field (discussed extensively at the 2018 International Complement Workshop in Santa Fe, NM).

The revised manuscript Discussion includes citations for the following landmark studies, and the pertinent findings are outlined below:

a) Claudia Kemper’s lab indicates that murine CD4 T cells lacking C3 have reduced Th1 responses (IFNγ) following in vitroexpansion and re-stimulation (Sup. Figure 4I in Liszewski et al., 2013).

– This paper’s primary finding is that C3a may have a critical survival function on human T cells, but this has not been recapitulated in mice.

– Similarly,Th1 responses are reduced in C3-deficient human T cells following in vitro stimulation (PMID: 24321396).

b) Peter Garred’s lab utilized human donor cells and found that C3 product/receptor expression were downregulated in T cells activated in vitro. They conclude that “endogenous[ly] expressed C3 is not vital for T cell turnover” (Hansen et al., 2019).

c) John Atkinson’s lab has shown that human CD4 T cells acquire C3 from the extracellular environment, but exogenous C3 did not alter IFNγ production upon activation in vitro (Elvington et al., 2017).

Importantly, the studies cited above investigate Th1 responses in human T cells with anti-CD3 and anti-CD46 stimulation in vitro. There are species-specific differences in complement biology between humans and mice:

a) While CD46 is required for Th1 polarization/cytokine production in human T cells, mice do not express CD46.

b) Instead, data from the Medof lab indicate that anaphylatoxin receptors are important for Th1 cytokine production in mice (Strainic et al., 2008).

In light of the studies listed above, we addressed several specific questions relative to C3 and T cell function using the HSV-1 ocular infection model as a way to prime T cells in vivo.

a) Our data show no difference in IFNγ production in T cells from C3^-/- and WT mice following in vivo priming by way of ocular HSV-1 infection (Figure 2C). This was determined using a PMA/ionomycin-based re-stimulation assay comparable to that used in the studies cited above.

b) Our data show that endogenous C3 production is not required for T cell functionality in mice. This is based on the ability of sorted C3^-/- T cells to mediate corneal sensation loss upon transfer into TCRα^-/- mice (Figure 2D).

c) The cornea contains high concentrations of C3 protein (Figure 5B) prior to T cell infiltration. Accordingly, there is little evidence that putative C3 production by murine T cells specifically tips the balance in favor of pathology.

d) It remains possible that anaphylatoxin receptor signaling from locally produced C3a (and potentially C5a generated downstream) modulates T cell effector function upon extravasation into the inflammatory milieu. However, anaphylatoxin receptor signaling was not investigated herein.

Finally, C3 breakdown products may lower the threshold for T cell activation/effector function through costimulatory pathways (e.g. CD46, C3aR1) akin to what has been shown for B cell responses through CD21 (PMID: 18328742, 30305631, 11418645, 25210145).

a) The clinical phenotype in the C3^-/- human population, albeit rare, involves high susceptibility to bacterial infections and immune complex-mediated diseases. However, published evidence suggesting that C3^-/- patients are more susceptible to viral infections is conspicuously absent (PMID: 16499568, 31192812, 18802120).

b) If a profound Th1 deficit existed in C3^-/- patients, their clinical phenotype would almost certainly include maladaptive responses to viral infections with increased morbidity.

c) Importantly, C3b and C4b are both ligands for CD46, thus C4b may compensate for the absence of C3b to facilitate Th1 induction in C3-deficient patients in vivo.

8) If C3 is predominant factor in causing the loss of corneal sensation in these models, what is the possible underlying mechanism?

The revised discussion now outlines a working hypothesis for the possible mechanism of corneal sensation loss (Discussion paragraph seven). This framework is a context-specific process involving C3 and CD4 T cells and is supported by established findings:

a) Corneal inflammation favors local activation of the complement pathway.

b) Sensory nerve terminals are damaged by sublytic MAC formation downstream of C3 activation (PMID: 21850539).

c) Cytokines produced by CD4 T cells (Th1) activate phagocytes (professional and nonprofessional) to consume damaged sensory nerve fibers.

d) Local complement activation may also augment Th1 cytokine secretion upon extravasation into the cornea via anaphlatoxin receptor signaling (PMID: 18328742).

The relevant complement pathways (classical, alternative, lectin, MAC-involvement), cellular targets, and the impact of anaphylatoxin receptor signaling are all topics of future investigation. By addressing the ‘heart’ of the complement pathway (C3), this study opens the door to future investigations to further elucidate the relevant pathomechanisms and corresponding therapeutic targets.

9) Can authors comment on the potential mechanisms of neuronal fiber retractions?

The nerve-intrinsic mechanism underlying sensory fiber retraction has not been clearly established for any corneal pathology to date. In line with the mechanism outlined above (critique #8), we hypothesize that membrane attack complex deposition on sensory fibers leads to damage and clearance. This is described in the revised manuscript:

“[An] imbalance in ‘complement proteostasis’ may favor deposition of complement fragments and sublytic MAC on corneal sensory nerve fibers (Tegla et al., 2011; Triantafilou et al., 2013). Notwithstanding, the nerve-intrinsic molecular mechanisms responsible for trigeminal sensory fiber retraction, neuronal death, or axonal regeneration in the cornea are largely unknown Stepp et al., 2017).”

Also, please see response to critique #6.

10) The use of CVF treatment offers a traditional approach to reduce complement activation. Given the developed therapies that target C5aR-signaling, authors should comment on the use of anti-C5a biologics for complement blockade in view of their discussion on potential therapies.

CVF was used merely as an experimental proof of concept. Subsequent studies are needed to investigate each complement initiation pathway, other effectors, and anaphylatoxin receptors.

Please see the “complement drug development pipeline” graphic featured in Ricklin et al., 2018.

There are many complement-targeted therapeutics in pre-clinical development or clinical trials (see the “complement drug development pipeline” graphic featured in Ricklin et al., 2018). The anti-C5 antibody Eculizumab is perhaps the most successful of the approved complement therapeutics, but its effect on C5aR signaling is indirect. In light of the numerous complement targets and concordant drugs in preclinical development, a particular focus on C5aR-signaling is hard to justify in the discussion, as our paper does not address C5 directly.

11) What possible factors from effector CD4 T cells are involved in activating C3 in GVHD and keratitis model?

Please see response to critique #8 above.

CD4 T cells are not required for C3 activation in the cornea. Our hypothesis is that T cell cytokines activate phagocytes to clear complement-tagged sensory nerve fibers.

This topic has been addressed in the revised Discussion:

“AED is driven primarily by a combined Th2/Th17 CD4 T cell response that differs from the Th1 bias observed in HSV-1 keratitis and ocular GHVD. This suggests that Th1 cytokines such as IFNγ, IL-2, and lymphotoxin-α may be important in coordinating corneal sensation loss.”

12) While AED seems to be a contrasting model, the authors find lack of sensory phenotypes and nerve loss in this model (Figure 6). Therefore, we do not think this is a good model as comparison. The limitations of comparing this vs. the other models should be Discussed.

Differences in CD4 T cell polarization among the models are now clearly highlighted in the Discussion (please see response 11).

To further address this point, we have added the following to the Discussion:

“The lack of sensory phenotype in the AED model was unanticipated, given that patients with severe allergy have been shown to have changes in nerve morphology… This contrasting model underscores the context-dependency of sensation loss during corneal inflammation.”

13) In Figure 1, it's interesting that while HSV-1 titers go up in the tear film and trigeminal ganglia (TG) at days 5 and 7 in C3^-/- mice compared to WT mice, HSV titers are not elevated in the C3^-/- cornea compared to WT mice. It's possible that the reason viral burdens are similar between WT and c3^-/- cornea is that a large number of nerve fibers and keratinocytes are destroyed that harbor the virus in WT mice. Could the authors address this point in the Discussion?

This topic has been addressed in the revised discussion in parallel with a commentary on the impact of HSV-1 glycoprotein C (Discussion section paragraph four).

Briefly, it is likely that the potent antiviral type-1 interferon response protects the cornea against excessive HSV-1 replication even in the absence of C3. Accordingly, the maintenance of corneal innervation in C3^-/- mice may enhance shedding of virus produced within the TG.

14) References: The authors can cite additional papers relevant to HSV-1 and complement C3: This includes the papers by Carroll and Knipe showing myeloid immune cell derived complement plays a role in HSV-1 host defense in peripheral tissues (Verschoor et al.,, 2001; Gadjeva et al., J Immunol. 2002 PMID: 12421924; Verschoor et al., J Immunol 2003 PMID:14607939) and papers showing that HSV-1 inhibits complement through glycoprotein C to evade host defense (Hook et al., J Virol 2006 PMID: 16571820; Lubinski et al., 2002)

In our model, corneal nerve damage occurs prior to generation of broad humoral immunity to HSV-1. The Carroll and Knipe papers cited above show the impact of bone marrow-derived complement on induction of humoral immunity to HSV-1, but they do not address host defense with respect to viral pathogenesis or tissue damage. The papers are solid, but their relationship to the current investigation is debatable.

However, the revised manuscript cites the Verschoor et al. paper (PMID: 11509581) as evidence of macrophage-derived C3 production and a 1999 PNAS paper from Knipe and Carroll corroborating normal proliferation of WT and C3^-/- T cells primed by vaccination with a replication defective virus based on [³H]thymidine uptake in response to viral antigen re-stimulation (PMID: 10535987).

The topic of glycoprotein C (gC) and complement evasion are now addressed in the Discussion (paragraph four).

Author details

1. Derek J Royer

   1. Department of Ophthalmology, Duke University Medical Center, Durham, United States
   2. Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, United States

   Present address

   Department of Ophthalmology, Duke Eye Center, Durham, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   Derek.Royer@Duke.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8476-1784

2. Jose Echegaray-Mendez

   Department of Ophthalmology, Duke University Medical Center, Durham, United States

   Contribution

   Resources, Investigation

   Competing interests

   No competing interests declared

3. Liwen Lin

   Department of Ophthalmology, Duke University Medical Center, Durham, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Grzegorz B Gmyrek

   Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Rose Mathew

   Department of Ophthalmology, Duke University Medical Center, Durham, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

6. Daniel R Saban

   1. Department of Ophthalmology, Duke University Medical Center, Durham, United States
   2. Department of Immunology, Duke University Medical Center, Durham, United States

   Contribution

   Supervision, Funding acquisition, Writing—review and editing

   Competing interests

   No competing interests declared

7. Victor L Perez

   Department of Ophthalmology, Duke University Medical Center, Durham, United States

   Contribution

   Supervision, Funding acquisition, Writing—review and editing

   Competing interests

   No competing interests declared

8. Daniel JJ Carr

   1. Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, United States
   2. Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, United States

   Contribution

   Supervision, Funding acquisition, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

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