Autoantibodies are associated with disease progression in idiopathic pulmonary fibrosis.

Several reports have highlighted a potential role of autoreactive B-cells and autoantibodies that correlates with increased disease severity in patients with idiopathic pulmonary fibrosis (IPF). Here we show that patients with IPF have an altered B-cell phenotype and that those subjects who have autoantibodies against the intermediate filament protein periplakin (PPL) have a significantly worse outcome in terms of progression-free survival. Using a mouse model of lung fibrosis, we demonstrate that introducing antibodies targeting the endogenous protein PPL (mimicking naturally occurring autoantibodies seen in patients) directly in the lung increases lung injury, inflammation, collagen and fibronectin expression through direct activation of follicular dendritic cells, which in turn activates and drives proliferation of fibroblasts. This fibrocyte population was also observed in fibrotic foci of patients with IPF and was increased in peripheral blood of IPF patients compared to aged-matched controls. This study reiterates the complex and heterogeneous nature of IPF, identifying new pathways that may prove suitable for therapeutic intervention.

Regulation of neutrophilic inflammation by proteinase-activated receptor 1 during bacterial pulmonary infection.

Neutrophils are key effector cells of the innate immune response to pathogenic bacteria, but excessive neutrophilic inflammation can be associated with bystander tissue damage. The mechanisms responsible for neutrophil recruitment to the lungs during bacterial pneumonia are poorly defined. In this study, we focus on the potential role of the major high-affinity thrombin receptor, proteinase-activated receptor 1 (PAR-1), during the development of pneumonia to the common lung pathogen Streptococcus pneumoniae. Our studies demonstrate that neutrophils were indispensable for controlling S. pneumoniae outgrowth but contributed to alveolar barrier disruption. We further report that intra-alveolar coagulation (bronchoalveolar lavage fluid thrombin-antithrombin complex levels) and PAR-1 immunostaining were increased in this model of bacterial lung infection. Functional studies using the most clinically advanced PAR-1 antagonist, SCH530348, revealed a key contribution for PAR-1 signaling in influencing neutrophil recruitment to lung airspaces in response to both an invasive and noninvasive strain of S. pneumoniae (D39 and EF3030) but that PAR-1 antagonism did not impair the ability of the host to control bacterial outgrowth. PAR-1 antagonist treatment significantly decreased pulmonary levels of IL-1ß, CXCL1, CCL2, and CCL7 and attenuated alveolar leak. Ab neutralization studies further demonstrated a nonredundant role for IL-1ß, CXCL1, and CCL7 in mediating neutrophil recruitment in response to S. pneumoniae infection. Taken together, these data demonstrate a key role for PAR-1 during S. pneumoniae lung infection that is mediated, at least in part, by influencing multiple downstream inflammatory mediators.

Regulation of neutrophilic inflammation in lung injury induced by community-acquired pneumonia.

BACKGROUND: Community-acquired pneumonia is commonly caused by Streptococcus pneumoniae, which is associated with excessive neutrophilic inflammation. The high-affinity thrombin receptor, proteinase-activated receptor 1 (PAR1), has been implicated in mediating the interplay between coagulation and inflammation. However, its role during S pneumoniae-induced neutrophilic inflammation, and the mechanisms for neutrophil recruitment in this context are poorly understood. We aimed to investigate the role of neutrophilic inflammation and PAR1 in S pneumoniae-induced pneumonia. METHODS: We used the most clinically advanced PAR-1 antagonist, SCH530348, and performed neutrophil depletion and chemokine neutralisation studies in two murine models. We also did translational studies to examine CXC and CC chemokine receptor expression by flow cytometry on neutrophils in blood and bronchoalveolar lavage fluid (BALF) from mechanically ventilated patients with acute respiratory distress syndrome induced by community-acquired pneumonia. FINDINGS: S pneumoniae infection led to activation of coagulation, increased neutrophil recruitment, and increased PAR-1 expression. By contrast with neutrophil depletion, PAR1 antagonist treatment significantly reduced neutrophil recruitment (mean difference 26·7 × 10(3) cells per mL [SE 4·9] at 4 h, p=0·0002; and 149·3 [41·4] at 24 h, p=0·0032) without being detrimental to host defence. Markers of alveolar leak, coagulation activation, and proinflammatory cytokines and chemokines (interleukin 1ß, CXCL1, CCL2, and CCL7) were attenuated. Neutralisation studies demonstrated that interleukin 1ß and CCL7, but not CXCL1 and CCL2, had a key role in neutrophil recruitment in this model. In patients with acute respiratory distress syndrome induced by community-acquired pneumonia (n=10), CXCR1 and CXCR2 expression on BALF neutrophils was higher than in controls (n=3) (median difference in mean fluorescence intensity [MFI] 703 arbitrary units [p=0·0699] for CXCR1 and 658·7 [p=0·0280] for CXCR2). The expression of CXCR1 was decreased on neutrophils from BALF compared with blood (median difference in MFI 1337, p=0·0020) and that of CXCR2, CCR1, CCR2, and CCR3 was increased (125·5, p=0·0020; 335·1, p=0·0020; 116, p=0·0068; and 275, p=0·0020; respectively). INTERPRETATION: These findings suggest that clinically available PAR1 antagonists might offer a novel therapeutic approach for prevention and management of excessive neutrophilic inflammation and alveolar barrier dysfunction in pneumococcal pneumonia without compromising host defence. Furthermore, these data highlight a role for chemokine receptor switching in acute respiratory distress syndrome induced by community-acquired pneumonia. FUNDING: Wellcome Trust.

Exploration of a potent PI3 kinase/mTOR inhibitor as a novel anti-fibrotic agent in IPF.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal of all fibrotic conditions with no curative therapies. Common pathomechanisms between IPF and cancer are increasingly recognised, including dysfunctional pan-PI3 kinase (PI3K) signalling as a driver of aberrant proliferative responses. GSK2126458 is a novel, potent, PI3K/mammalian target of rapamycin (mTOR) inhibitor which has recently completed phase I trials in the oncology setting. Our aim was to establish a scientific and dosing framework for PI3K inhibition with this agent in IPF at a clinically developable dose. METHODS: We explored evidence for pathway signalling in IPF lung tissue and examined the potency of GSK2126458 in fibroblast functional assays and precision-cut IPF lung tissue. We further explored the potential of IPF patient-derived bronchoalveolar lavage (BAL) cells to serve as pharmacodynamic biosensors to monitor GSK2126458 target engagement within the lung. RESULTS: We provide evidence for PI3K pathway activation in fibrotic foci, the cardinal lesions in IPF. GSK2126458 inhibited PI3K signalling and functional responses in IPF-derived lung fibroblasts, inhibiting Akt phosphorylation in IPF lung tissue and BAL derived cells with comparable potency. Integration of these data with GSK2126458 pharmacokinetic data from clinical trials in cancer enabled modelling of an optimal dosing regimen for patients with IPF. CONCLUSIONS: Our data define PI3K as a promising therapeutic target in IPF and provide a scientific and dosing framework for progressing GSK2126458 to clinical testing in this disease setting. A proof-of-mechanism trial of this agent is currently underway. TRIAL REGISTRATION NUMBER: NCT01725139, pre-clinical.

Evidence for a functional thymic stromal lymphopoietin signaling axis in fibrotic lung disease.

Thymic stromal lymphopoietin (TSLP) recently has emerged as a key cytokine in the development of type 2 immune responses. Although traditionally associated with allergic inflammation, type 2 responses are also recognized to contribute to the pathogenesis of tissue fibrosis. However, the role of TSLP in the development of non-allergen-driven diseases, characterized by profibrotic type 2 immune phenotypes and excessive fibroblast activation, remains underexplored. Fibroblasts represent the key effector cells responsible for extracellular matrix production but additionally play important immunoregulatory roles, including choreographing immune cell recruitment through chemokine regulation. The aim of this study was to examine whether TSLP may be involved in the pathogenesis of a proto-typical fibrotic disease, idiopathic pulmonary fibrosis (IPF). We combined the immunohistochemical analysis of human IPF biopsy material with signaling studies by using cultured primary human lung fibroblasts and report for the first time, to our knowledge, that TSLP and its receptor (TSLPR) are highly upregulated in IPF. We further show that lung fibroblasts represent both a novel cellular source and target of TSLP and that TSLP induces fibroblast CCL2 release (via STAT3) and subsequent monocyte chemotaxis. These studies extend our understanding of TSLP as a master regulator of type 2 immune responses beyond that of allergic inflammatory conditions and suggest a novel role for TSLP in the context of chronic fibrotic lung disease.

Proteinase-activated receptor-1, CCL2, and CCL7 regulate acute neutrophilic lung inflammation.

PAR1 plays a central role in mediating the interplay between coagulation and inflammation, but its role in regulating acute neutrophilic inflammation is unknown. We report that antagonism of PAR1 was highly effective at reducing acute neutrophil accumulation in a mouse model of LPS-induced lung inflammation. PAR1 antagonism also reduced alveolar-capillary barrier disruption in these mice. This protection was associated with a reduction in the expression of the chemokines, CCL2 and CCL7, but not the proinflammatory cytokines, TNF and IL-6, or the classic neutrophil chemoattractants, CXCL1 and CXCL2. Antibody neutralization of CCL2 and CCL7 significantly reduced LPS-induced total leukocyte and neutrophil accumulation, recovered from the bronchoalveolar lavage fluid of challenged mice. Immunohistochemical analysis revealed that CCL2 predominantly localized to alveolar macrophages and pulmonary epithelial cells, whereas CCL7 was restricted to the pulmonary epithelium. In keeping with these observations, the intranasal administration of recombinant CCL2 (rCCL2) and rCCL7 led to the accumulation of neutrophils within the lung airspaces of naive mice in the absence of any underlying inflammation. Flow cytometry analysis further demonstrated an increase in Ly6G(hi) neutrophils expressing the chemokine receptors, CCR1 and CCR2, isolated from mouse lungs compared with circulating neutrophils. Conversely, the expression of CXCR2 decreased on neutrophils isolated from the lung compared with circulating neutrophils. Furthermore, this switch in chemokine receptor expression was accentuated after acute LPS-induced lung inflammation. Collectively, these findings reveal a novel role for PAR1 and the chemokines, CCL2 and CCL7, during the early events of acute neutrophilic inflammation.

AZD3152 neutralizes SARS-CoV-2 historical and contemporary variants and is protective in hamsters and well tolerated in adults.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in variants that can escape neutralization by therapeutic antibodies. Here, we describe AZD3152, a SARS-CoV-2-neutralizing monoclonal antibody designed to provide improved potency and coverage against emerging variants. AZD3152 binds to the back left shoulder of the SARS-CoV-2 spike protein receptor binding domain and prevents interaction with the human angiotensin-converting enzyme 2 receptor. AZD3152 potently neutralized a broad panel of pseudovirus variants, including the currently dominant Omicron variant JN.1 but has reduced potency against XBB subvariants containing F456L. In vitro studies confirmed F456L resistance and additionally identified T415I and K458E as escape mutations. In a Syrian hamster challenge model, prophylactic administration of AZD3152 protected hamsters from weight loss and inflammation-related lung pathologies and reduced lung viral load. In the phase 1 sentinel safety cohort of the ongoing SUPERNOVA study (ClinicalTrials.gov: NCT05648110), a single 600-mg intramuscular injection of AZD5156 (containing 300 mg each of AZD3152 and cilgavimab) was well tolerated in adults through day 91. Observed serum concentrations of AZD3152 through day 91 were similar to those observed with cilgavimab and consistent with predictions for AZD7442, a SARS-CoV-2-neutralizing antibody combination of cilgavimab and tixagevimab, in a population pharmacokinetic model. On the basis of its pharmacokinetic characteristics, AZD3152 is predicted to provide durable protection against symptomatic coronavirus disease 2019 caused by susceptible SARS-CoV-2 variants, such as JN.1, in humans.

Multiparametric MRI of early tumor response to immune checkpoint blockade in metastatic melanoma.

BACKGROUND: Immune checkpoint inhibitors are now standard of care treatment for many cancers. Treatment failure in metastatic melanoma is often due to tumor heterogeneity, which is not easily captured by conventional CT or tumor biopsy. The aim of this prospective study was to investigate early microstructural and functional changes within melanoma metastases following immune checkpoint blockade using multiparametric MRI. METHODS: Fifteen treatment-naïve metastatic melanoma patients (total 27 measurable target lesions) were imaged at baseline and following 3 and 12 weeks of treatment on immune checkpoint inhibitors using: T2-weighted imaging, diffusion kurtosis imaging, and dynamic contrast-enhanced MRI. Treatment timepoint changes in tumor cellularity, vascularity, and heterogeneity within individual metastases were evaluated and correlated to the clinical outcome in each patient based on Response Evaluation Criteria in Solid Tumors V.1.1 at 1 year. RESULTS: Differential tumor growth kinetics in response to immune checkpoint blockade were measured in individual metastases within the same patient, demonstrating significant intertumoral heterogeneity in some patients. Early detection of tumor cell death or cell loss measured by a significant increase in the apparent diffusivity (Dapp) (p<0.05) was observed in both responding and pseudoprogressive lesions after 3 weeks of treatment. Tumor heterogeneity, as measured by apparent diffusional kurtosis (Kapp), was consistently higher in the pseudoprogressive and true progressive lesions, compared with the responding lesions throughout the first 12 weeks of treatment. These preceded tumor regression and significant tumor vascularity changes (Ktrans, ve, and vp) detected after 12 weeks of immunotherapy (p<0.05). CONCLUSIONS: Multiparametric MRI demonstrated potential for early detection of successful response to immune checkpoint inhibitors in metastatic melanoma.

Intratumoral IL12 mRNA Therapy Promotes TH1 Transformation of the Tumor Microenvironment.

PURPOSE: While immune checkpoint inhibitors such as anti-PD-L1 are rapidly becoming the standard of care in the treatment of many cancers, only a subset of treated patients have long-term responses. IL12 promotes antitumor immunity in mouse models; however, systemic recombinant IL12 had significant toxicity and limited efficacy in early clinical trials. EXPERIMENTAL DESIGN: We therefore designed a novel intratumoral IL12 mRNA therapy to promote local IL12 tumor production while mitigating systemic effects. RESULTS: A single intratumoral dose of mouse (m)IL12 mRNA induced IFNγ and CD8+ T-cell-dependent tumor regression in multiple syngeneic mouse models, and animals with a complete response demonstrated immunity to rechallenge. Antitumor activity of mIL12 mRNA did not require NK and NKT cells. mIL12 mRNA antitumor activity correlated with TH1 tumor microenvironment (TME) transformation. In a PD-L1 blockade monotherapy-resistant model, antitumor immunity induced by mIL12 mRNA was enhanced by anti-PD-L1. mIL12 mRNA also drove regression of uninjected distal lesions, and anti-PD-L1 potentiated this response. Importantly, intratumoral delivery of mRNA encoding membrane-tethered mIL12 also drove rejection of uninjected lesions with very limited circulating IL12p70, supporting the hypothesis that local IL12 could induce a systemic antitumor immune response against distal lesions. Furthermore, in ex vivo patient tumor slice cultures, human IL12 mRNA (MEDI1191) induced dose-dependent IL12 production, downstream IFNγ expression and TH1 gene expression. CONCLUSIONS: These data demonstrate the potential for intratumorally delivered IL12 mRNA to promote TH1 TME transformation and robust antitumor immunity.See related commentary by Cirella et al., p. 6080.

Pancreatic ß-Cell Rest Replenishes Insulin Secretory Capacity and Attenuates Diabetes in an Extreme Model of Obese Type 2 Diabetes.

The onset of common obesity-linked type 2 diabetes (T2D) is marked by exhaustive failure of pancreatic ß-cell functional mass to compensate for insulin resistance and increased metabolic demand, leading to uncontrolled hyperglycemia. Here, the ß-cell-deficient obese hyperglycemic/hyperinsulinemic KS db/db mouse model was used to assess consequential effects on ß-cell functional recovery by lowering glucose homeostasis and/or improving insulin sensitivity after treatment with thiazolidinedione therapy or glucagon-like peptide 1 receptor agonism alone or in combination with sodium/glucose cotransporter 2 inhibition (SGLT-2i). SGLT-2i combination therapies improved glucose homeostasis, independent of changes in body weight, resulting in a synergistic increase in pancreatic insulin content marked by significant recovery of the ß-cell mature insulin secretory population but with limited changes in ß-cell mass and no indication of ß-cell dedifferentiation. Restoration of ß-cell insulin secretory capacity also restored biphasic insulin secretion. These data emphasize that by therapeutically alleviating the demand for insulin in vivo, irrespective of weight loss, endogenous ß-cells recover significant function that can contribute to attenuating diabetes. Thus, this study provides evidence that alleviation of metabolic demand on the ß-cell, rather than targeting the ß-cell itself, could be effective in delaying the progression of T2D.

Transformation of the tumour microenvironment by a CD40 agonist antibody correlates with improved responses to PD-L1 blockade in a mouse orthotopic pancreatic tumour model.

Despite the availability of recently developed chemotherapy regimens, survival times for pancreatic cancer patients remain poor. These patients also respond poorly to immune checkpoint blockade therapies (anti-CTLA-4, anti-PD-L1, anti-PD-1), which suggests the presence of additional immunosuppressive mechanisms in the pancreatic tumour microenvironment (TME). CD40 agonist antibodies (αCD40) promote antigen presenting cell (APC) maturation and enhance macrophage tumouricidal activity, and may therefore alter the pancreatic TME to increase sensitivity to immune checkpoint blockade. Here, we test whether αCD40 transforms the TME in a mouse syngeneic orthotopic model of pancreatic cancer, to increase sensitivity to PD-L1 blockade. We found that whilst mice bearing orthotopic Pan02 tumours responded poorly to PD-L1 blockade, αCD40 improved overall survival. αCD40 transformed the TME, upregulating Th1 chemokines, increasing cytotoxic T cell infiltration and promoting formation of an immune cell-rich capsule separating the tumour from the normal pancreas. Furthermore, αCD40 drove systemic APC maturation, memory T cell expansion, and upregulated tumour and systemic PD-L1 expression. Combining αCD40 with PD-L1 blockade enhanced anti-tumour immunity and improved overall survival versus either monotherapy. These data provide further support for the potential of combining αCD40 with immune checkpoint blockade to promote anti-tumour immunity in pancreatic cancer.