IL-36, which belongs to the IL-1 superfamily, is increasingly linked to neutrophilic inflammation. Here, we combined in vivo and in vitro approaches using primary mouse and human cells, as well as, acute and chronic mouse models of lung inflammation to provide mechanistic insight into the intercellular signaling pathways and mechanisms through which IL-36 promotes lung inflammation. IL-36 receptor deficient mice exposed to cigarette smoke or cigarette smoke and H1N1 influenza virus had attenuated lung inflammation compared with wild-type controls. We identified neutrophils as a source of IL-36 and show that IL-36 is a key upstream amplifier of lung inflammation by promoting activation of neutrophils, macrophages and fibroblasts through cooperation with GM-CSF and the viral mimic poly(I:C). Our data implicate IL-36, independent of other IL-1 family members, as a key upstream amplifier of neutrophilic lung inflammation, providing a rationale for targeting IL-36 to improve treatment of a variety of neutrophilic lung diseases.

Anti-TNF therapies are a core anti-inflammatory approach for chronic diseases such as rheumatoid arthritis and Crohn’s Disease. Previously, we and others found that TNF blocks the emergence and function of alternative-activated or M2 macrophages involved in wound healing and tissue-reparative functions. Conceivably, anti-TNF drugs could mediate their protective effects in part by an altered balance of macrophage activity. To understand the mechanistic basis of how TNF regulates tissue-reparative macrophages, we used RNAseq, scRNAseq, ATACseq, time-resolved phospho-proteomics, gene-specific approaches, metabolic analysis, and signaling pathway deconvolution. We found that TNF controls tissue-reparative macrophage gene expression in a highly gene-specific way, dependent on JNK signaling via the type 1 TNF receptor on specific populations of alternative-activated macrophages. We further determined that JNK signaling has a profound and broad effect on activated macrophage gene expression. Our findings suggest that TNF’s anti-M2 effects evolved to specifically modulate components of tissue and reparative M2 macrophages and TNF is therefore a context-specific modulator of M2 macrophages rather than a pan-M2 inhibitor.

Current treatments fail to modify the underlying pathophysiology and disease progression of chronic obstructive pulmonary disease (COPD), necessitating alternative therapies. Here, we show that COPD subjects have increased IL-36γ and decreased IL-36 receptor antagonist (IL-36Ra) in bronchoalveolar and nasal fluid compared to control subjects. IL-36γ is derived from small airway epithelial cells (SAEC) and further induced by a viral mimetic, whereas IL-36RA is derived from macrophages. IL-36γ stimulates release of the neutrophil chemoattractants CXCL1 and CXCL8, as well as elastolytic matrix metalloproteinases (MMPs) from small airway fibroblasts (SAF). Proteases released from COPD neutrophils cleave and activate IL-36γ thereby perpetuating IL-36 inflammation. Transfer of culture media from SAEC to SAF stimulated release of CXCL1, that was inhibited by exogenous IL-36RA. The use of a therapeutic antibody that inhibits binding to the IL-36 receptor (IL-36R) attenuated IL-36γ driven inflammation and cellular cross talk. We have demonstrated a mechanism for the amplification and propagation of neutrophilic inflammation in COPD and that blocking this cytokine family via a IL-36R neutralizing antibody could be a promising new therapeutic strategy in the treatment of COPD.

Idiopathic Pulmonary Fibrosis (IPF) is characterized by the aberrant deposition and organization of extracellular matrix (ECM). Increased stiffness of fibrotic lung tissue is a major contributor to fibrosis by perpetuating fibrotic responses, through modulation of interactions between ECM-producing cells and fibrotic ECM. Human decellularized lung ECM-derived hydrogels can be used as a model for mimicking the native three-dimensional (3D) microenvironment, including recapitulating the mechanical environment in nonfibrotic (control) and fibrotic lung tissues. In this study, we aimed to characterize control and fibrotic human lung ECM-derived 3D hydrogels seeded with primary lung fibroblasts to investigate the cellular remodeling responses dictated by the origin of the microenvironment. IPF and control decellularized lung matrices were freeze-dried, ground to a fine powder, and mixed (pool of 7 donors per diagnosis) before pepsin digestion. Primary lung fibroblasts were isolated from lung tissue of patients with IPF (IPF) or macroscopically normal lung tissue derived from patients undergoing tumor resection (non-IPF). IPF or non-IPF lung-derived fibroblasts (n=6 each) were resuspended in pH-neutralized ECM solutions and hydrogels were allowed to form before being cultured at 37°C, 5% CO 2 . Cell-seeded ECM-derived hydrogels were harvested on day 14 and their stiffness was measured using a Low-Load Compression Tester at 20% strain and compared with equivalent cell-free hydrogels. Cell-free IPF hydrogels were stiffer than cell-free control hydrogels on day 14. Time in culture did not result in a difference in stiffness of cellfree hydrogels. The stiffness of control hydrogels seeded with either IPF or non-IPF fibroblasts did not change over a 14-day culture period (Figure 1A, 1B). In contrast, the stiffness of IPF hydrogels was increased in the presence of non-IPF lung fibroblasts, compared to cell-free hydrogels over the 14 day period (Figure 1C) (p = 0.016). However, when IPF fibroblasts were seeded in IPF hydrogels the stiffness of the hydrogel did not change compared to the cell-free counterpart (Figure 1D). These results illustrate the importance of both the fibroblast-origin and the origin of the ECM in determining the response of the fibroblasts to the 3D ECM microenvironment. Specifically, the fibrotic microenvironment evoked a profibrotic response in non-IPF fibroblasts while IPF fibroblasts did not exhibit the same response in this microenvironment. Taken together, our data have implications for cellular therapies that rely on adding non-diseased cells to tissues to promote disease resolution, as well as revealing the complexity of the positive feedback between fibrotic ECM and fibroblasts.