Complement regulation in the eye: implications for age-related macular degeneration.

Careful regulation of the complement system is critical for enabling complement proteins to titrate immune defense while also preventing collateral tissue damage from poorly controlled inflammation. In the eye, this balance between complement activity and inhibition is crucial, as a low level of basal complement activity is necessary to support ocular immune privilege, a prerequisite for maintaining vision. Dysregulated complement activation contributes to parainflammation, a low level of inflammation triggered by cellular damage that functions to reestablish homeostasis, or outright inflammation that disrupts the visual axis. Complement dysregulation has been implicated in many ocular diseases, including glaucoma, diabetic retinopathy, and age-related macular degeneration (AMD). In the last two decades, complement activity has been the focus of intense investigation in AMD pathogenesis, leading to the development of novel therapeutics for the treatment of atrophic AMD. This Review outlines recent advances and challenges, highlighting therapeutic approaches that have advanced to clinical trials, as well as providing a general overview of the complement system in the posterior segment of the eye and selected ocular diseases.

Microbiota produced indole metabolites disrupt host cell mitochondrial energy production and inhibit Cryptosporidium parvum growth.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children in resource-poor settings. Susceptibility rapidly declines with age, associated with changes in the microbiota. To explore microbial influences on susceptibility, we screened 85 microbiota- associated metabolites enriched in the adult gut for their effects on C. parvum growth in vitro. We identified eight inhibitory metabolites in three main classes: secondary bile salts/acids, a vitamin B 6 precursor, and indoles. Growth restriction of C. parvum by indoles did not depend on the host aryl hydrocarbon receptor (AhR) pathway. Instead, treatment impaired host mitochondrial function and reduced total cellular ATP, as well as directly reduced the membrane potential in the parasite mitosome, a degenerate mitochondria. Oral administration of indoles, or reconstitution of the gut microbiota with indole producing bacteria, delayed life cycle progression of the parasite in vitro and reduced severity of C. parvum infection in mice. Collectively, these findings indicate that microbiota metabolites contribute to colonization resistance to Cryptosporidium infection.

Microbiota-produced indole metabolites disrupt mitochondrial function and inhibit Cryptosporidium parvum growth.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children in resource-poor settings. To explore microbial influences on susceptibility, we screened 85 microbiota-associated metabolites for their effects on Cryptosporidium parvum growth in vitro. We identify eight inhibitory metabolites in three main classes: secondary bile salts/acids, a vitamin B6 precursor, and indoles. Growth restriction of C. parvum by indoles does not depend on the host aryl hydrocarbon receptor (AhR) pathway. Instead, treatment impairs host mitochondrial function and reduces total cellular ATP, as well as directly reducing the membrane potential in the parasite mitosome, a degenerate mitochondria. Oral administration of indoles, or reconstitution of the gut microbiota with indole-producing bacteria, delays life cycle progression of the parasite in vitro and reduces the severity of C. parvum infection in mice. Collectively, these findings indicate that microbiota metabolites impair mitochondrial function and contribute to colonization resistance to Cryptosporidium infection.

Commensal Cryptosporidium colonization elicits a cDC1-dependent Th1 response that promotes intestinal homeostasis and limits other infections.

Cryptosporidium can cause severe diarrhea and morbidity, but many infections are asymptomatic. Here, we studied the immune response to a commensal strain of Cryptosporidium tyzzeri (Ct-STL) serendipitously discovered when conventional type 1 dendritic cell (cDC1)-deficient mice developed cryptosporidiosis. Ct-STL was vertically transmitted without negative health effects in wild-type mice. Yet, Ct-STL provoked profound changes in the intestinal immune system, including induction of an IFN-γ-producing Th1 response. TCR sequencing coupled with in vitro and in vivo analysis of common Th1 TCRs revealed that Ct-STL elicited a dominant antigen-specific Th1 response. In contrast, deficiency in cDC1s skewed the Ct-STL CD4 T cell response toward Th17 and regulatory T cells. Although Ct-STL predominantly colonized the small intestine, colon Th1 responses were enhanced and associated with protection against Citrobacter rodentium infection and exacerbation of dextran sodium sulfate and anti-IL10R-triggered colitis. Thus, Ct-STL represents a commensal pathobiont that elicits Th1-mediated intestinal homeostasis that may reflect asymptomatic human Cryptosporidium infection.

In Vitro Culture of Cryptosporidium parvum Using Stem Cell-Derived Intestinal Epithelial Monolayers.

Cryptosporidium parvum has a complex life cycle consisting of asexual and sexual phases that culminate in oocyst formation in vivo. The most widely used cell culture platforms to study C. parvum only support a few days of growth and do not allow the parasite to proceed past the sexual stages to complete oocyst formation. Additionally, these cell culture platforms are mostly adenocarcinoma cell lines, which do not adequately model the parasite's natural environment in the small intestine. We describe here a method to create primary intestinal epithelial cell monolayers that support long-term C. parvum growth. Monolayers were derived from mouse intestinal stem cells grown as spheroids and plated onto transwells, allowing for separate apical and basolateral compartments. In the apical chamber, the cell growth medium was removed to create an "air-liquid interface" that enhanced host cell differentiation and supported long-term C. parvum growth. The use of primary intestinal cells to grow C. parvum in vitro will be a valuable tool for studying host-parasite interactions using a convenient in vitro model that more closely resembles the natural niche in the intestine.

A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability.

Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile experimental methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using "air-liquid interface" (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable completion of the parasite life cycle in vitro. ALI cultures support parasite expansion > 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions.

Monoclonal Antibodies to Intracellular Stages of Cryptosporidium parvum Define Life Cycle Progression In Vitro.

Among the obstacles hindering Cryptosporidium research is the lack of an in vitro culture system that supports complete life development and propagation. This major barrier has led to a shortage of widely available anti-Cryptosporidium antibodies and a lack of markers for staging developmental progression. Previously developed antibodies against Cryptosporidium were raised against extracellular stages or recombinant proteins, leading to antibodies with limited reactivity across the parasite life cycle. Here we sought to create antibodies that recognize novel epitopes that could be used to define intracellular development. We identified a mouse epithelial cell line that supported C. parvum growth, enabling immunization of mice with infected cells to create a bank of monoclonal antibodies (MAbs) against intracellular parasite stages while avoiding the development of host-specific antibodies. From this bank, we identified 12 antibodies with a range of reactivities across the parasite life cycle. Importantly, we identified specific MAbs that can distinguish different life cycle stages, such as trophozoites, merozoites, type I versus II meronts, and macrogamonts. These MAbs provide valuable tools for the Cryptosporidium research community and will facilitate future investigation into parasite biology.IMPORTANCECryptosporidium is a protozoan parasite that causes gastrointestinal disease in humans and animals. Currently, there is a limited array of antibodies available against the parasite, which hinders imaging studies and makes it difficult to visualize the parasite life cycle in different culture systems. In order to alleviate this reagent gap, we created a library of novel antibodies against the intracellular life cycle stages of Cryptosporidium We identified antibodies that recognize specific life cycle stages in distinctive ways, enabling unambiguous description of the parasite life cycle. These MAbs will aid future investigation into Cryptosporidium biology and help illuminate growth differences between various culture platforms.

Persistent reversal of enhanced amphetamine intake by transient CaMKII inhibition.

Amphetamine exposure transiently increases Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) α expression in the nucleus accumbens (NAcc) shell and this persistently increases local GluA1 S831 phosphorylation and enhances behavioral responding to the drug. Here we assessed whether transiently interfering with CaMKII signaling using a dominant-negative CaMKIIα mutant delivered to the NAcc shell with herpes simplex viral vectors could reverse these long-lasting biochemical and behavioral effects observed following exposure to amphetamine. As expected, transient expression of CaMKIIα K42M in the NAcc shell produced a corresponding transient increase in CaMKIIα and decrease in pCaMKIIα (T286) protein levels in this site. Remarkably, this transient inhibition of CaMKII activity produced a long-lasting reversal of the increased GluA1 S831 phosphorylation levels in NAcc shell and persistently blocked the enhanced locomotor response to and self-administration of amphetamine normally observed in rats previously exposed to the drug. Together, these results indicate that even transient interference with CaMKII signaling may confer long-lasting benefits in drug-sensitized individuals and point to CaMKII and its downstream pathways as attractive therapeutic targets for the treatment of stimulant addiction.

A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice.

Staphylococcus aureus is a major cause of human disease, responsible for half a million infections and approximately 20,000 deaths per year in the United States alone. This pathogen secretes α-hemolysin, a pore-forming cytotoxin that contributes to the pathogenesis of pneumonia. α-hemolysin injures epithelial cells in vitro by interacting with its receptor, the zinc-dependent metalloprotease ADAM10 (ref. 6). We show here that mice harboring a conditional disruption of the Adam10 gene in lung epithelium are resistant to lethal pneumonia. Investigation of the molecular mechanism of toxin-receptor function revealed that α-hemolysin upregulates ADAM10 metalloprotease activity in alveolar epithelial cells, resulting in cleavage of the adherens junction protein E-cadherin. Cleavage is associated with disruption of epithelial barrier function, contributing to the pathogenesis of lethal acute lung injury. A metalloprotease inhibitor of ADAM10 prevents E-cadherin cleavage in response to Hla; similarly, toxin-dependent E-cadherin proteolysis and barrier disruption is attenuated in ADAM10-knockout mice. Together, these data attest to the function of ADAM10 as the cellular receptor for α-hemolysin. The observation that α-hemolysin can usurp the metalloprotease activity of its receptor reveals a previously unknown mechanism of pore-forming cytotoxin action in which pathologic insults are not solely the result of irreversible membrane injury and defines ADAM10 inhibition as a strategy to attenuate α-hemolysin-induced disease.

Role of a disintegrin and metalloprotease 10 in Staphylococcus aureus alpha-hemolysin-mediated cellular injury.

Staphylococcus aureus alpha-hemolysin (Hla), a potent cytotoxin, plays an important role in the pathogenesis of staphylococcal diseases, including those caused by methicillin-resistant epidemic strains. Hla is secreted as a water-soluble monomer that undergoes a series of conformational changes to generate a heptameric, beta-barrel structure in host membranes. Structural maturation of Hla depends on its interaction with a previously unknown proteinaceous receptor in the context of the cell membrane. It is reported here that a disintegrin and metalloprotease 10 (ADAM10) interacts with Hla and is required to initiate the sequence of events whereby the toxin is transformed into a cytolytic pore. Hla binding to the eukaryotic cell requires ADAM10 expression. Further, ADAM10 is required for Hla-mediated cytotoxicity, most notably when the toxin is present at low concentrations. These data thus implicate ADAM10 as the probable high-affinity toxin receptor. Upon Hla binding, ADAM10 relocalizes to caveolin 1-enriched lipid rafts that serve as a platform for the clustering of signaling molecules. It is demonstrated that the Hla-ADAM10 complex initiates intracellular signaling events that culminate in the disruption of focal adhesions.

Transient overexpression of alpha-Ca2+/calmodulin-dependent protein kinase II in the nucleus accumbens shell enhances behavioral responding to amphetamine.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is known to contribute to the expression of psychostimulant sensitization by regulating dopamine (DA) overflow from DA neuron terminals in the nucleus accumbens (NAcc). The present experiments explored the contribution of CaMKII in NAcc neurons postsynaptic to these terminals where it is known to participate in a number of signaling pathways that regulate responding to psychostimulant drugs. Exposure to amphetamine transiently increased alphaCaMKII levels in the shell but not the core of the NAcc. Thus, HSV (herpes simplex viral) vectors were used to transiently overexpress alphaCaMKII in NAcc neurons in drug-naive rats, and behavioral responding to amphetamine was assessed. Transiently overexpressing alphaCaMKII in the NAcc shell led to long-lasting enhancement of amphetamine-induced locomotion and self-administration manifested when alphaCaMKII levels were elevated and persisting long after they had returned to baseline. Enhanced locomotion was not observed after infection in the NAcc core or sites adjacent to the NAcc. Transient elevation of NAcc shell alphaCaMKII levels also enhanced locomotor responding to NAcc AMPA and increased phosphorylation levels of GluR1 (Ser831), a CaMKII site, both soon and long after infection. Similar increases in pGluR1 (Ser831) were observed both soon and long after exposure to amphetamine. These results indicate that the transient increase in alphaCaMKII observed in neurons of the NAcc shell after viral-mediated gene transfer and likely exposure to amphetamine leads to neuroadaptations in AMPA receptor signaling in this site that may contribute to the long-lasting maintenance of behavioral and incentive sensitization by psychostimulant drugs like amphetamine.