Therapies capable of resolving rheumatoid arthritis (RA)-associated lung disease, particularly in the setting of environmental exposures, are lacking. This study sought to determine whether blocking interleukin (IL)-33 in a murine model of RA-associated lung disease induced by combined collagen-induced arthritis (CIA) and repeated inhalant lipopolysaccharide (LPS) exposures could be advantageous. Arthritis prone male DBA/1J mice received CIA (vs saline) injections (days 1 and 21) plus intranasal LPS (100 ng) or saline daily for 4 weeks and were subsequently treated with 5 doses of anti-IL-33 (10 mg/kg) or isotype over 11 days. Anti-IL-33 therapy significantly (P < .05) mitigated several CIA+LPS-induced effects. Anti-IL-33 reduced arthritis inflammatory score by 76% and serum acute phase reactant pentraxin-2 by 70% and reversed weight loss induced by CIA+LPS coexposure. Anti-IL-33 reduced CIA+LPS-induced antimalondialdehyde acetaldehyde but not anticitrullinated autoantibodies in serum and reduced serum levels of inflammatory mediators. Lung cell infiltrates induced by coexposure, including neutrophils, activated lung macrophages, transitional monocytes-macrophages, monocytic-like cells, and dendritic cells, were reduced with anti-IL-33. Anti-IL-33 also reduced CIA+LPS-induced lung levels of chemokines involved in monocyte-macrophage recruitment and fibrotic/repair mediators collagen deposition, IL-33 expression, inflammatory CC motif chemokine receptor 2+ monocytes/macrophages, and airway Muc5b immunostaining induced by CIA+LPS were reduced with anti-IL-33. CIA+LPS-induced lung expression of post-translational modified autoantigens including citrullinated and malondialdehyde acetaldehyde and its common substrate, vimentin, was reduced with anti-IL-33 treatment. Blocking IL-33 was beneficial in hastening lung recovery after establishment of disease by coexposure to CIA+LPS and potentially represents a novel therapeutic approach in RA-associated lung disease. Significance Statement Blocking the interleukin (IL)-33 signaling pathway after the establishment of, but not during the induction of, rheumatoid arthritis (RA)-associated lung disease, as modeled by coexposure of collagen-induced arthritis with repetitive inhalant endotoxin, demonstrated beneficial consequences in reducing inflammatory joint, systemic, and lung disease. Treatment with anti-IL-33 agents, particularly in men with a history of environmental exposures, may represent a novel approach to reduce disease burden in humans with established RA-lung disease, underscoring an important role for the IL-33 pathway.

Abstract Background Parasitic nematodes have evolved secreted effector proteins which interact with host molecules to modulate the host immune response. These interactions can be highly specific and are under strong selective pressure. Here, we investigate the host-pathogen interaction between Heligmosomoides bakeri and its natural host Mus musculus , and between H. polygyrus and its natural host Apodemus sylvaticus . We focus on the Alarmin Release Inhibitor family (previously called HpARI) produced by heligmosomid parasites, which interact with IL-33 and heparan sulphate. Results Using comparative genomics, we identified duplications and diversifications of the ARI family including multiple copies of ARI1 and conserved ARI3 homologues in both H. bakeri and H. polygyrus. We identify a new family member, HpolARI4, encoded by H. polygyrus, as well as a more distant homologue, HmixARI, encoded by Heligmosomum mixtum, a parasite of the bank vole ( Myodes glareolus). Our functional assays identify that HpolARI4 preferentially blocks A. sylvaticus IL-33 but has little activity against M. musculus IL-33, whereas HbakARI2 is highly specific for M. musculus IL-33. We furthermore show differential interaction with host heparan sulphate, with HbakARI2 and HpolARI4 showing high affinity binding, whereas HbakARI3 shows no binding. We further test this family against non-host human IL-33 and show that functional suppression can be achieved by HbakARI3 in vitro and in vivo . Conclusion By integrating genomic data, structural insights, and functional assays, we reveal how the expansion and diversification of the ARI family drive host-specific IL-33 modulation, providing a molecular model for nematode-host co-evolution.

RATIONALE:Bronchiolitis is the commonest cause of hospital admission in children under the age of 1 year, most cases being due to respiratory syncytial virus (RSV) infection. The mechanisms causing infantile bronchiolitis are incompletely understood but include a deficient mucosal interferon response, neutrophilic inflammation and enhanced mucosal Type-2 responses. OBJECTIVES:We sought to determine the mucosal immune processes associated with severe paediatric bronchiolitis. METHODS:We performed transcriptomic analyses on mucosal samples from infants hospitalized with Moderate (n = 48) and Severe (n = 40) bronchiolitis. Differential expression and regression analyses determined genes associated with different severity categories. Responses were modelled in vitro using air-liquid interface human nasal epithelial cell culture models. MEASUREMENTS AND MAIN RESULTS:We confirmed weakened interferon-associated signalling in severe RSV and non-RSV bronchiolitis but unexpectedly found elevated IL-36α (an IL-1 family cytokine implicated in chronic inflammatory diseases) early in infection. Conversely, IL36A was decreased in whole blood during severe RSV, suggesting that this association is unique to the mucosa. In human nasal epithelial cells grown in vitro under air-liquid interface we found IL-36α to be produced by epithelial cells during RSV infection and that its secretion is enhanced by neutrophils. CONCLUSIONS:These findings implicate mucosal IL-36α as a dominant feature of severe paediatric bronchiolitis. ONE SENTENCE SUMMARY:Mucosal transcriptomics identifies IL-36 secretion as a feature of life-threatening paediatric RSV and all-cause bronchiolitis associated with interferon response failure.

Advancements in single-cell technologies and deep sequencing have expanded the B cell repertoire available for antibody discovery. However, selecting the highest-affinity antibodies from many sequences remains challenging, reflecting our incomplete understanding of the mechanisms sustaining affinity maturation and associated molecular markers. Here, we generated datasets of antigen-specific B cells after mouse immunization and reanalyzed public data to identify "High Signature" (HS), a transcriptomic signature predictive of high-affinity antibodies. HS was derived through differential expression analyses and machine learning by integrating antibody sequences, gene expression, and affinity measurements of expressed antibodies. HS enabled sub-nanomolar-affinity antibody selection without prior sequence analysis in de novo immunization campaigns. HS-expressing B cells were 3 times more likely to yield high-affinity antibodies than randomly picked cells. HS demonstrated translatability to two human PBMC datasets from COVID patients, resulting in enriched high-affinity antibody selection, highlighting its antibody discovery potential across species. A record of this paper's transparent peer review process is included in the supplemental information.

Rationale Information is missing on the tissue cell expression patterns of interleukin 33 (IL-33) and splice variants of the IL-33 receptor ST2 in normal and chronic obstructive pulmonary disease (COPD) lungs.Objectives To characterize the expression patterns of IL-33, the soluble ST2 (sST2) and membrane-bound ST2 (ST2L) splice variants in the poorly studied small airway and distal lung compartments in COPD and controls.Methods Surgically excised lung tissue was collected from 38 COPD patients and 21 non-COPD controls. Lung compartment expression of IL-33 and ST2 and key expressing cell types were assessed histologically by combined in situ hybridization and multiplex immunohistochemistry. Expression dynamics of IL-33, ST2L, and sST2 were explored by spatially resolved single-cell analysis.Measurements and Main Results COPD lungs displayed increased IL-33 mRNA and IL-33 mRNA/protein ratios, suggesting increased IL-33 turnover. Total ST2/IL1RL1 mRNA levels were upregulated in COPD lungs. Mast cells constituted the major ST2-expressing immune cell population in controls and displayed a microenvironmental-specific upregulation of both ST2L and sST2 in COPD. In control alveolar regions, ST2Lhigh sST2high mast cells were present alongside IL-33-expressing general capillary (gCap) and sST2moderate ST2Llow aerocyte endothelial subsets. In COPD, patchy alveolar regions displayed markedly elevated capillary sST2 and numbers of ST2L+ and IL-33+ gCaps.Conclusions By unraveling the expression patterns of IL-33 and the biologically opposing ST2L and sST2 splice variants in control and COPD lungs, the present study provides novel insights into IL-33-mediated immunity in the distal lung, information that has bearing on treatment strategies targeting this pathway in lung diseases.

Tozorakimab is a human monoclonal antibody that neutralizes interleukin (IL)-33. IL-33 is a broad-acting epithelial "alarmin" cytokine upregulated in lung tissue of patients with chronic obstructive pulmonary disease (COPD). This first-in-human, phase I, randomized, double-blind, placebo-controlled study (NCT03096795) evaluated the safety, tolerability, pharmacokinetics (PKs), immunogenicity, target engagement, and pharmacodynamics (PDs) of tozorakimab. This was a 3-part study. In part 1, 56 healthy participants with a history of mild atopy received single escalating doses of either intravenous or subcutaneous tozorakimab or placebo. In part 2, 24 patients with mild COPD received multiple ascending doses of subcutaneous tozorakimab or placebo. In part 3, 8 healthy Japanese participants received a single intravenous dose of tozorakimab or placebo. The safety data collected included treatment-emergent adverse events (TEAEs), vital signs, and clinical laboratory parameters. Biological samples for PKs, immunogenicity, target engagement, and PD biomarker analyses were collected. No meaningful differences in the frequencies of TEAEs were observed between the tozorakimab and placebo arms. Three tozorakimab-treated participants with COPD experienced treatment-emergent serious adverse events. Subcutaneous or intravenous tozorakimab demonstrated linear, time-independent PKs with a mean half-life of 11.7-17.3 days. Treatment-emergent anti-drug antibody frequency was low. Engagement of tozorakimab with endogenous IL-33 in serum and nasal airways was demonstrated. Tozorakimab significantly reduced serum IL-5 and IL-13 levels in patients with COPD compared with placebo. Overall, tozorakimab was well tolerated, with a linear, time-independent serum PK profile. Additionally, biomarker studies demonstrated proof of mechanism. Overall, these data support the further clinical development of tozorakimab in COPD and other inflammatory diseases.

Interleukin (IL)-33 is released following tissue damage, causing airway inflammation and remodelling via reduced IL-33 (IL-33red)/serum stimulation-2 (ST2) and oxidised IL-33 (IL-33ox)/receptor for advanced glycation end products (RAGE)/epidermal growth factor receptor (EGFR) pathways. This study aimed to identify associations of IL-33 with clinical outcomes and pathological mechanisms during viral lower respiratory tract disease (LRTD). Ultra-sensitive immunoassays were developed to measure IL-33red, IL-33ox and IL-33/sST2 complexes in samples from patients hospitalised with COVID-19. Immunohistochemistry and multiomics were used to characterise lung samples. Elevated IL-33 in the airway and IL-33/sST2 complex in the circulation correlated with poor clinical outcomes (death, need for intensive care or mechanical ventilation). IL-33 was localised to airway epithelial and endothelial barriers, whereas IL1RL1 was expressed on aerocytes, alveolar endothelial cells specialised for gaseous exchange. IL-33 increased expression of mediators of neutrophilic inflammation, immune cell infiltration, interferon signalling and coagulation in endothelial cell cultures. Endothelial IL-33 signatures were strongly related with signatures associated with viral LRTD. Increased IL-33 release following respiratory viral infections is associated with poor clinical outcomes and might contribute to alveolar dysfunction. Although this does not show a causal relationship with disease, these results provide a rationale to evaluate pathological roles for IL-33 in viral LRTD.

AimsThis study describes the pharmacokinetic (PK)/target engagement (TE) relationship of tozorakimab, an anti-interleukin (IL)-33 antibody, by building a mechanistic population PK/TE model using phase 1 biomarker data.MethodsThe analysis included tozorakimab PK and TE in serum assessed in 60 tozorakimab-treated participants, including healthy adults and patients with mild chronic obstructive pulmonary disease. Scenarios evaluated three dose frequencies (once every 2, 4 or 6 weeks) administered subcutaneously at seven doses of tozorakimab (30, 60, 90, 120, 150, 300 or 600 mg). For each dose, simulations were performed with 5000 virtual individuals to predict systemic TE. Inhibition of IL-33/soluble ST2 (sST2) complex levels at trough PK at steady state was assessed in each dosing scenario. The PK/TE modelling analyses were performed using a nonlinear mixed-effect modelling approach.ResultsThe final two-compartment PK model with tozorakimab binding IL-33 in the central compartment adequately described the systemic PK and TE of tozorakimab at population and individual levels. The mean PK parameter estimates of absorption rate, central volume of distribution and clearance were 0.48 (90% confidence interval [CI]: 0.40-0.59, 1/day), 12.64 (90% CI: 8.60-18.62, L) and 0.87 (90% CI: 0.65-1.16, L/day), respectively. Consistent with the observed value, tozorakimab bioavailability was 45%. For all three dose frequencies, predicted inhibition of systemic IL-33/sST2 levels was more than 95% at doses greater than 90 mg.ConclusionsThe PK/TE model reliably quantified the relationship between PK and systemic TE of tozorakimab, with potential utility for predicting clinical dose-response relationships and supporting clinical dose selection.

Tozorakimab is a human monoclonal antibody that neutralizes interleukin (IL)-33. IL-33 is a broad-acting epithelial "alarmin" cytokine upregulated in lung tissue of patients with chronic obstructive pulmonary disease (COPD). This first-in-human, phase I, randomized, double-blind, placebo-controlled study (NCT03096795) evaluated the safety, tolerability, pharmacokinetics (PKs), immunogenicity, target engagement, and pharmacodynamics (PDs) of tozorakimab. This was a 3-part study. In part 1, 56 healthy participants with a history of mild atopy received single escalating doses of either intravenous or subcutaneous tozorakimab or placebo. In part 2, 24 patients with mild COPD received multiple ascending doses of subcutaneous tozorakimab or placebo. In part 3, 8 healthy Japanese participants received a single intravenous dose of tozorakimab or placebo. The safety data collected included treatment-emergent adverse events (TEAEs), vital signs, and clinical laboratory parameters. Biological samples for PKs, immunogenicity, target engagement, and PD biomarker analyses were collected. No meaningful differences in the frequencies of TEAEs were observed between the tozorakimab and placebo arms. Three tozorakimab-treated participants with COPD experienced treatment-emergent serious adverse events. Subcutaneous or intravenous tozorakimab demonstrated linear, time-independent PKs with a mean half-life of 11.7-17.3 days. Treatment-emergent anti-drug antibody frequency was low. Engagement of tozorakimab with endogenous IL-33 in serum and nasal airways was demonstrated. Tozorakimab significantly reduced serum IL-5 and IL-13 levels in patients with COPD compared with placebo. Overall, tozorakimab was well tolerated, with a linear, time-independent serum PK profile. Additionally, biomarker studies demonstrated proof of mechanism. Overall, these data support the further clinical development of tozorakimab in COPD and other inflammatory diseases.

Idiopathic Pulmonary Fibrosis (IPF) is a devastating form of respiratory disease with a life expectancy of 3–4 years. Inflammation, epithelial injury and myofibroblast proliferation have been implicated in disease initiation and, recently, epithelial-fibroblastic crosstalk has been identified as a central driver. However, the ability to interrogate this crosstalk is limited due to the absence of in vitro models that mimic physiological conditions. To investigate IPF dysregulated cross-talk, primary normal human bronchial epithelial (NHBE) cells and primary normal human lung fibroblasts (NHLF) or diseased human lung fibroblasts (DHLF) from IPF patients, were co-cultured in direct contact at the air–liquid interface (ALI). Intercellular crosstalk was assessed by comparing cellular phenotypes of co-cultures to respective monocultures, through optical, biomolecular and electrical methods. A co-culture-dependent decrease in epithelium thickness, basal cell mRNA (P63, KRT5) and an increase in transepithelial electrical resistance (TEER) was observed. This effect was significantly enhanced in DHLF co-cultures and lead to the induction of epithelial to mesenchymal transition (EMT) and increased mRNA expression of TGFβ-2, ZO-1 and DN12. When stimulated with exogenous TGFβ, NHBE and NHLF monocultures showed a significant upregulation of EMT (COL1A1, FN1, VIM, ASMA) and senescence (P21) markers, respectively. In contrast, direct NHLF/NHBE co-culture indicated a protective role of epithelial-fibroblastic cross-talk against TGFβ-induced EMT, fibroblast-to-myofibroblast transition (FMT) and inflammatory cytokine release (IL-6, IL-8, IL-13, IL-1β, TNF-α). DHLF co-cultures showed no significant phenotypic transition upon stimulation, likely due to the constitutively high expression of TGFβ isoforms prior to any exogenous stimulation. The model developed provides an alternative method to generate IPF-related bronchial epithelial phenotypes in vitro, through the direct co-culture of human lung fibroblasts with NHBEs. These findings highlight the importance of fibroblast TGFβ signaling in EMT but that monocultures give rise to differential responses compared to co-cultures, when exposed to this pro-inflammatory stimulus. This holds implications for any translation conclusions drawn from monoculture studies and is an important step in development of more biomimetic models of IPF. In summary, we believe this in vitro system to study fibroblast-epithelial crosstalk, within the context of IPF, provides a platform which will aid in the identification and validation of novel targets.

Interleukin (IL)-33 is a broad-acting alarmin cytokine that can drive inflammatory responses following tissue damage or infection and is a promising target for treatment of inflammatory disease. Here, we describe the identification of tozorakimab (MEDI3506), a potent, human anti-IL-33 monoclonal antibody, which can inhibit reduced IL-33 (IL-33 red ) and oxidized IL-33 (IL-33 ox ) activities through distinct serum-stimulated 2 (ST2) and receptor for advanced glycation end products - epidermal growth factor receptor (RAGE-EGFR complex) signalling pathways. We hypothesized that a therapeutic antibody would require an affinity higher than that of ST2 for IL-33, with an association rate greater than 10 7 M −1 s −1 , to effectively neutralize IL-33 following rapid release from damaged tissue. An innovative antibody generation campaign identified tozorakimab, an antibody with a femtomolar affinity for IL-33 red and a fast association rate (8.5 × 10 7 M −1 s −1 ), which was comparable to soluble ST2. Tozorakimab potently inhibited ST2-dependent inflammatory responses driven by IL-33 in primary human cells and in a murine model of lung epithelial injury. Additionally, tozorakimab prevented the oxidation of IL-33 and its activity via the RAGE/EGFR signalling pathway, thus increasing in vitro epithelial cell migration and repair. Tozorakimab is a novel therapeutic agent with a dual mechanism of action that blocks IL-33 red and IL-33 ox signalling, offering potential to reduce inflammation and epithelial dysfunction in human disease.

Compared to intramuscular vaccines, nasally administered vaccines have the advantage of inducing local mucosal immune responses that may block infection and interrupt transmission of respiratory pathogens. Live attenuated influenza vaccine (LAIV) is effective in preventing influenza in children, but a correlate of protection for LAIV remains unclear. Studying young adult volunteers, we observe that LAIV induces distinct, compartmentalized, antibody responses in the mucosa and blood. Seeking immunologic correlates of these distinct antibody responses we find associations with mucosal IL-33 release in the first 8 hours post-inoculation and divergent CD8 + and circulating T follicular helper (cTfh) T cell responses 7 days post-inoculation. Mucosal antibodies are induced separately from blood antibodies, are associated with distinct immune responses early post-inoculation, and may provide a correlate of protection for mucosal vaccination. This study was registered as NCT04110366 and reports primary (mucosal antibody) and secondary (blood antibody, and nasal viral load and cytokine) endpoint data.

The cytokine, interleukin-33 (IL-33), has emerged as a promising therapeutic target in chronic respiratory diseases and viral exacerbations. Despite great progress being made with simple cell assays, aspects of IL-33 biology require further investigation in complex, translational systems. Consequently, the aim of this work was to develop a human precision-cut lung slice (huPCLS) platform suitable to explore IL-33 biology and test novel therapies. huPCLS were generated and cultured for up to 7 days. LDH levels and H&E staining were used to evaluate tissue viability and architecture with inflammatory cytokine release following LPS stimulation measured to assess huPCLS responsiveness. IL-33 stimulated huPCLS were analysed via bulk RNA-sequencing (RNA-seq) whilst immunohistochemistry was used to identify airway epithelial subsets. huPCLS were also exposed to human rhinovirus A (HRV-A) with cytokine release utilised as a readout of infection. LDH levels peaked 1 day post slicing and decreased with time in culture. Tissue architecture was maintained at day 7 versus day 0 whilst 24-hour LPS stimulation induced significant IL-6, IL-1β and TNFα release relative to unstimulated huPCLS. In pilot studies, IL-33 responsive genes were upregulated when IL-33-stimulated huPCLS were analysed by bulk RNA-seq. Furthermore, airway epithelial subsets known to be modulated by IL-33 could be identified in huPCLS. Finally, infection with HRV-A increased IFNγ and IL-5 release by huPCLS relative to control slices. In summary, a method to generate viable and responsive huPCLS has been developed with promising initial results demonstrating the potential of the platform to investigate IL-33 biology and screen novel therapies.

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease (ILD) with limited treatment options. Interleukin-33 (IL-33) is proposed to play a role in the development of IPF however the exclusive use of prophylactic dosing regimens means that the therapeutic benefit of targeting this cytokine in IPF is unclear. IL-33 expression was assessed in ILD lung sections and human lung fibroblasts (HLFs) by immunohistochemistry and gene/protein expression and responses of HLFs to IL-33 stimulation measured by qPCR. In vivo, the fibrotic potential of IL-33:ST2 signalling was assessed using a murine model of bleomycin (BLM)-induced pulmonary fibrosis and therapeutic dosing with an ST2-Fc fusion protein. Lung and bronchoalveolar lavage fluid were collected for measurement of inflammatory and fibrotic endpoints. Human precision-cut lung slices (PCLS) were stimulated with transforming growth factor-β (TGFβ) or IL-33 and fibrotic readouts assessed. IL-33 was expressed by fibrotic fibroblasts in situ and was increased by TGFβ treatment in vitro. IL-33 treatment of HLFs did not induce IL6, CXCL8, ACTA2 and COL1A1 mRNA expression with these cells found to lack the IL-33 receptor ST2. Similarly, IL-33 stimulation had no effect on ACTA2, COL1A1, FN1 and fibronectin expression by PCLS. Despite having effects on inflammation suggestive of target engagement, therapeutic dosing with the ST2-Fc fusion protein failed to reduce BLM-induced fibrosis measured by hydroxyproline content or Ashcroft score. Together these findings suggest the IL-33:ST2 axis does not play a central fibrogenic role in the lungs with therapeutic blockade of this pathway unlikely to surpass the current standard of care for IPF.