Luke Pennington

Structural basis of omalizumab therapy and omalizumab-mediated IgE exchange

Omalizumab is a widely used therapeutic anti-IgE antibody. Here we report the crystal structure of the omalizumab–Fab in complex with an IgE-Fc fragment. This structure reveals the mechanism of omalizumab-mediated inhibition of IgE interactions with both high- and low-affinity IgE receptors, and explains why omalizumab selectively binds free IgE. The structure of the complex also provides mechanistic insight into a class of disruptive IgE inhibitors that accelerate the dissociation of the high-affinity IgE receptor from IgE. We use this structural data to generate a mutant IgE-Fc fragment that is resistant to omalizumab binding. Treatment with this omalizumab-resistant IgE-Fc fragment, in combination with omalizumab, promotes the exchange of cell-bound full-length IgE with omalizumab-resistant IgE-Fc fragments on human basophils. This combination treatment also blocks basophil activation more efficiently than either agent alone, providing a novel approach to probe regulatory mechanisms underlying IgE hypersensitivity with implications for therapeutic interventions.

H-IPSE Is a Pathogen-Secreted Host Nucleus-Infiltrating Protein (Infiltrin) Expressed Exclusively by the Schistosoma haematobium Egg Stage

ABSTRACT Urogenital schistosomiasis, caused by the parasitic trematode Schistosoma haematobium, affects over 112 million people worldwide. As with Schistosoma mansoni infections, the pathology of urogenital schistosomiasis is related mainly to the egg stage, which induces granulomatous inflammation of affected tissues. Schistosoma eggs and their secretions have been studied extensively for the related organism S. mansoni, which is more amenable to laboratory studies. Indeed, we have shown that IPSE/alpha-1 (here M-IPSE), a major protein secreted from S. mansoni eggs, can infiltrate host cells. Although the function of M-IPSE is unknown, its ability to translocate to the nuclei of host cells and bind DNA suggests a possible role in immune modulation of host cell tissues. Whether IPSE homologs are expressed in other schistosome species has not been investigated. Here, we describe the cloning of two paralog genes, H03-IPSE and H06-IPSE, which are orthologs of M-IPSE, from egg cDNA of S. haematobium. Using PCR and immunodetection, we confirmed that the expression of these genes is restricted to the egg stage and female adult worms, while the H-IPSE protein is detectable only in mature eggs and not adults. We show that both H03-IPSE and H06-IPSE proteins can infiltrate HTB-9 bladder cells when added exogenously to culture medium. Monopartite C-terminal nuclear localization sequence (NLS) motifs conserved in H03-IPSE, SKRRRKY, and H06-IPSE SKRGRKY, are responsible for targeting the proteins to the nucleus of HTB-9 cells, as demonstrated by site-directed mutagenesis and green fluorescent protein (GFP) tagging. Thus, S. haematobium eggs express IPSE homologs that appear to perform similar functions in infiltrating host cells.

A new mouse model for female genital schistosomiasis.

Background Over 112 million people worldwide are infected with Schistosoma haematobium, one of the most prevalent schistosome species affecting humans. Female genital schistosomiasis (FGS) occurs when S. haematobium eggs are deposited into the female reproductive tract by adult worms, which can lead to pelvic pain, vaginal bleeding, genital disfigurement and infertility. Recent evidence suggests co-infection with S. haematobium increases the risks of contracting sexually transmitted diseases such as HIV. The associated mechanisms remain unclear due to the lack of a tractable animal model. We sought to create a mouse model conducive to the study of immune modulation and genitourinary changes that occur with FGS. Methods To model FGS in mice, we injected S. haematobium eggs into the posterior vaginal walls of 30 female BALB/c mice. A control group of 20 female BALB/c mice were injected with uninfected LVG hamster tissue extract. Histology, flow cytometry and serum cytokine levels were assessed at 2, 4, 6, and 8 weeks post egg injection. Voiding studies were performed at 1 week post egg injection. Results Vaginal wall injection with S. haematobium eggs resulted in synchronous vaginal granuloma development within 2 weeks post-egg injection that persisted for at least 6 additional weeks. Flow cytometric analysis of vaginal granulomata revealed infiltration by CD4+ T cells with variable expression of the HIV co-receptors CXCR4 and CCR5. Granulomata also contained CD11b+F4/80+ cells (macrophages and eosinophils) as well as CXCR4+MerTK+ macrophages. Strikingly, vaginal wall-injected mice featured significant urinary frequency despite the posterior vagina being anatomically distant from the bladder. This may represent a previously unrecognized overactive bladder response to deposition of schistosome eggs in the vagina. Conclusion We have established a new mouse model that could potentially enable novel studies of genital schistosomiasis in females. Ongoing studies will further explore the mechanisms by which HIV target cells may be drawn into FGS-associated vaginal granulomata.

Therapeutic exploitation of IPSE, a urogenital parasite-derived host modulatory protein, for chemotherapy-induced hemorrhagic cystitis

Chemotherapy‐induced hemorrhagic cystitis (CHC) can be difficult to manage. Prior work suggests that IL‐4 alleviates ifosfamide‐induced hemorrhagic cystitis (IHC), but systemically administered IL‐4 causes significant side effects. We hypothesized that the Schistosoma hematobium homolog of IL‐4‐inducing principle from Schistosoma mansoni eggs (H‐IPSE), would reduce IHC and associated bladder pathology. IPSE binds IgE on basophils and mast cells, triggering IL‐4 secretion by these cells. IPSE is also an “infiltrin,” translocating into the host nucleus to modulate gene transcription. Mice were administered IL‐4, H‐IPSE protein or its nuclear localization sequence (NLS) mutant, with or without neutralizing anti‐IL‐4 antibody, or 2‐mercaptoethane sulfonate sodium (MESNA; a drug used to prevent IHC), followed by ifosfamide. Bladder tissue damage and hemoglobin content were measured. Spontaneous and evoked pain, urinary frequency, and bladdergene expression analysis were assessed. Pain behaviors were interpreted in a blinded fashion. One dose of H‐IPSE was superior to MESNA and IL‐4 in suppressing bladder hemorrhage in an IL‐4‐dependent fashion and comparable with MESNA in dampening ifosfamide‐triggered pain behaviors in an NLS‐dependent manner. H‐IPSE also accelerated urothelial repair following IHC. Our work represents the first therapeutic exploitation of a uropathogen‐derived host modulatory molecule in a clinically relevant bladder disease model and indicates that IPSE may be an alternative to MESNA for mitigating CHC.—Mbanefo, E. C., Le, L., Pennington, L. F., Odegaard, J. I., Jardetzky, T. S., Alouffi, A., Falcone, F. H., Hsieh, M. H. Therapeutic exploitation of IPSE, a urogenital parasite‐derived host modulatory protein, for chemotherapy‐induced hemorrhagic cystitis. FASEB J. 32, 4408–4419 (2018). www.fasebj.org

IPSE, a urogenital parasite-derived immunomodulatory protein, ameliorates ifosfamide-induced hemorrhagic cystitis through downregulation of pro-inflammatory pathways

Ifosfamide and other oxazaphosphorines can result in hemorrhagic cystitis, a constellation of complications caused by acrolein metabolites. We previously showed that a single dose of IPSE, a schistosome-derived host modulatory protein, can ameliorate ifosfamide-related cystitis; however, the exact mechanisms underlying this urotoxic effect and its prevention are not fully understood. To provide insights into IPSE’s protective mechanism, we undertook transcriptional profiling of bladders from ifosfamide-treated mice, with or without IPSE pretreatment. Following ifosfamide challenge, there was upregulation of a range of pro-inflammatory genes. The pro-inflammatory pathway involving the IL-1β, TNFα and IL-6 triad via NFkB and STAT3 signaling pathways was identified as the key driver of inflammation. The NRF2-mediated oxidative stress response pathway, which regulates both Hmox1-mediated heme homoeostasis and expression of antioxidant enzymes, was highly activated. Anti-inflammatory and cellular proliferation cascades implicated in tissue repair, namely Wnt, Hedgehog and PPAR pathways, were downregulated. IPSE administration before ifosfamide injection resulted in significant downregulation of major proinflammatory pathways including the triad of IL-1β, TNFα and IL-6 pathways, the interferon signaling pathway, and less apparent reduction in oxidative stress responses. Taken together, we have identified signatures of acute phase inflammation and oxidative stress responses in the ifosfamide-injured bladder, which are reversed by pretreatment with IPSE, a parasite derived anti-inflammatory molecule. In addition to providing new insights into the underlying mechanism of IPSE’s therapeutic effects, this work has revealed several pathways that could be therapeutically targeted to prevent and treat ifosfamide-induced hemorrhagic cystitis.

IPSE, a parasite-derived host immunomodulatory protein, is a promising therapeutic for hemorrhagic cystitis

Chemotherapy-induced hemorrhagic cystitis is characterized by bladder pain and voiding dysfunction caused by hemorrhage and inflammation. Of currently available therapies, prophylactic 2-mercaptoethanesulfonic acid (MESNA) has limited efficacy and cannot treat pre-existing lesions. Therefore, novel therapeutic options to treat hemorrhagic cystitis are needed. We previously reported that systemic administration of the Schistosomiasis haematobium-derived protein H-IPSEH06 (IL-4-inducing principle from Schistosoma mansoni eggs), is superior to 3 doses of MESNA in alleviating hemorrhagic cystitis. Based on prior reports by others on S. mansoni IPSE and additional work by our group, we reasoned that H-IPSEH06 mediates its effects on hemorrhagic cystitis by binding IgE on basophils and inducing IL-4 expression, promoting urothelial proliferation, and translocating to the nucleus to modulate expression of genes implicated in relieving bladder dysfunction. We speculated that local bladder injection of the S. haematobium IPSE ortholog IPSEH03, hereafter called H-IPSEH03, might be more efficacious in preventing hemorrhagic cystitis compared to systemic administration of IPSEH06. We demonstrate herein that H-IPSEH03 is a promising therapeutic for the treatment of voiding dysfunction and bladder pain in hemorrhagic cystitis. Namely, it attenuates ifosfamide-induced increases in bladder wet weight in an IL-4-dependent fashion. H-IPSEH03 relieves hemorrhagic cystitis-associated allodynia. Finally, H-IPSEH03 drives increased urothelial cell proliferation. This indicates that IPSE induces bladder healing mechanisms, which suggests that it may be a novel non-opioid analgesic to treat bladder pain syndromes.

The soluble isoform of human FcɛRI is an endogenous inhibitor of IgE-mediated mast cell responses

The soluble isoform of FcɛRI, the high‐affinity IgE receptor (sFcεRI), is a protein of the IgE network with poorly defined functions.

MP29-06 THERAPEUTIC EXPLOITATION OF THE SCHISTOSOMA HAEMATOBIUM HOMOLOG OF INTERLEUKIN-4-INDUCING PRINCIPLE OF SCHISTOSOMA MANSONI EGGS FOR CHEMOTHERAPY-INDUCED HEMORRHAGIC CYSTITIS AND BLADDER HYPERSENSITIVITY

INTRODUCTION AND OBJECTIVES: Ifosfamide-induced hemorrhagic cystitis and bladder hypersensitivity can be difficult to manage when mesna fails to prevent them. Bladder hypersensitivity associated with various forms of cystitis may be refractory to multiple treatment modalities. Prior work suggests interleukin-4 (IL4) alleviates ifosfamide-induced hemorrhagic cystitis and resiniferatoxin (capsaicin receptor agonist)-induced bladder pain. IL4-inducing principle of Schistosoma mansoni eggs (IPSE) is a host modulatory protein that binds immunoglobulins on leukocytes thereby inducing IL4 production and translocates into host nuclei to alter gene transcription. We sought to determine if the S. haematobium homolog of IPSE (H-IPSE) would reduce ifosfamideand resiniferatoxin-induced bladder pathology. METHODS: We cloned and expressed H-IPSE and a nuclear localization sequence (NLS)-deficient mutant H-IPSE (H-IPSE[NLS]). H-IPSE IgE binding was measured by ELISA. H-IPSE activation of IgEbearing basophils was assayed using RSATL8 basophilic reporter cells. Cellular uptake and NLS-dependent nuclear translocation of H-IPSE and H-IPSE(NLS) were confirmed using HTB9 urothelial cells and fluorescence microscopy. We administered IL4, H-IPSE, H-IPSE+antiIL4 antibody, H-IPSE(NLS), or H-IPSE(NLS)+anti-IL4 antibody to mice prior to ifosfamide (with and without mesna) or resiniferatoxin. Negative controls were administered saline only. Positive controls were administered ifosfamide only. Previously published metrics for pain and urinary frequency were interpreted in blinded fashion. Bladder histology was interpreted in blinded fashion. Bladder hemoglobin was quantified using Drabkin0s assay. Bladder gene expression was assessed via realtime PCR. RESULTS: H-IPSE bound IgE in vitro and activated IgE-bearing RSATL8 cells. Nuclear translocation of H-IPSE but not H-IPSE(NLS) was confirmed. H-IPSE was superior to mesna and IL4 in suppressing ifosfamide-induced bladder hemorrhage (IL4-dependent). H-IPSE was comparable to mesna in dampening ifosfamide-triggered pain behaviors (NLS-dependent) and urinary frequency (NLS-dependent). H-IPSE reduced resiniferatoxin-mediated freezing behaviors (IL4and NLSdependent). H-IPSE reduced mRNA expression of proinflammatory mediators and increased expression of uroplakin mRNA. CONCLUSIONS: Our work suggests a uropathogen-derived host modulatory protein has therapeutic effects in bladder disease models.