MAPK13 controls structural remodeling and disease after epithelial injury.

All living organisms are charged with repair after injury particularly at epithelial barrier sites, but in some cases this response leads instead to structural remodeling and long-term disease. Identifying the molecular and cellular control of this divergence is key to disease modification. In that regard, stress kinase control of epithelial stem cells is a rational entry point for study. Here we examine the potential for mitogen-activated protein kinase 13 (MAPK13) regulation of epithelial stem cells using models of respiratory viral injury and post-viral lung disease. We show that Mapk13 gene-knockout mice handle acute infectious illness as expected but are protected against structural remodeling manifest as basal-epithelial stem cell (basal-ESC) hyperplasia-metaplasia, immune activation, and mucinous differentiation. In corresponding cell models, Mapk13-deficiency directly attenuates basal-ESC growth and organoid formation. Extension to human studies shows marked induction/activation of basal-cell MAPK13 in clinical samples of comparable remodeling found in asthma and COPD. Here again, MAPK13 gene-knockdown inhibits human basal-ESC growth in culture. Together, the data identify MAPK13 as a control for structural remodeling and disease after epithelial injury and as a suitable target for down-regulation as a disease-modifying strategy.

A potent MAPK13-14 inhibitor prevents airway inflammation and mucus production.

Common respiratory diseases continue to represent a major public health problem, and much of the morbidity and mortality is due to airway inflammation and mucus production. Previous studies indicated a role for mitogen-activated protein kinase 14 (MAPK14) in this type of disease, but clinical trials are unsuccessful to date. Our previous work identified a related but distinct kinase known as MAPK13 that is activated in respiratory airway diseases and is required for mucus production in human cell-culture models. Support for MAPK13 function in these models came from effectiveness of MAPK13 versus MAPK14 gene-knockdown and from first-generation MAPK13-14 inhibitors. However, these first-generation inhibitors were incompletely optimized for blocking activity and were untested in vivo. Here we report the next generation and selection of a potent MAPK13-14 inhibitor (designated NuP-3) that more effectively downregulates type-2 cytokine-stimulated mucus production in air-liquid interface and organoid cultures of human airway epithelial cells. We also show that NuP-3 treatment prevents respiratory airway inflammation and mucus production in new minipig models of airway disease triggered by type-2 cytokine challenge or respiratory viral infection. The results thereby provide the next advance in developing a small-molecule kinase inhibitor to address key features of respiratory disease.NEW & NOTEWORTHY This study describes the discovery of a potent mitogen-activated protein kinase 13-14 (MAPK13-14) inhibitor and its effectiveness in models of respiratory airway disease. The findings thereby provide a scheme for pathogenesis and therapy of lung diseases [e.g., asthma, chronic obstructive pulmonary disease (COPD), Covid-19, postviral, and allergic respiratory disease] and related conditions that implicate MAPK13-14 function. The findings also refine a hypothesis for epithelial and immune cell functions in respiratory disease that features MAPK13 as a possible component of this disease process.

A potent MAPK13-14 inhibitor prevents airway inflammation and mucus production.

Common respiratory diseases continue to represent a major public health problem, and much of the morbidity and mortality is due to airway inflammation and mucus production. Previous studies indicated a role for mitogen-activated protein kinase 14 (MAPK14) in this type of disease, but clinical trials are unsuccessful to date. Our previous work identified a related but distinct kinase known as MAPK13 that is activated in respiratory airway diseases and is required for mucus production in human cell-culture models. Support for MAPK13 function in these models came from effectiveness of MAPK13 versus MAPK14 gene-knockdown and from first-generation MAPK13-14 inhibitors. However, these first-generation inhibitors were incompletely optimized for blocking activity and were untested in vivo. Here we report the next generation and selection of a potent MAPK13-14 inhibitor (designated NuP-3) that more effectively down-regulates type-2 cytokine-stimulated mucus production in air-liquid interface and organoid cultures of human airway epithelial cells. We also show that NuP-3 treatment prevents respiratory airway inflammation and mucus production in new minipig models of airway disease triggered by type-2 cytokine challenge or respiratory viral infection. The results thereby provide the next advance in developing a small-molecule kinase inhibitor to address key features of respiratory disease.

Chloride channel accessory 1 gene deficiency causes selective loss of mucus production in a new pig model.

Morbidity and mortality of respiratory diseases are linked to airway obstruction by mucus but there are still no specific, safe, and effective drugs to correct this phenotype. The need for better treatment requires a new understanding of the basis for mucus production. In that regard, studies of human airway epithelial cells in primary culture show that a mucin granule constituent known as chloride channel accessory 1 (CLCA1) is required for inducible expression of the inflammatory mucin MUC5AC in response to potent type 2 cytokines. However, it remained uncertain whether CLCLA1 is necessary for mucus production in vivo. Conventional approaches to functional biology using targeted gene knockout were difficult due to the functional redundancy of additional Clca genes in mice not found in humans. We reasoned that CLCA1 function might be better addressed in pigs that maintain the same four-member CLCA gene locus and the corresponding mucosal and submucosal populations of mucous cells found in humans. Here we develop to our knowledge the first CLCA1-gene-deficient (CLCA1-/-) pig and show that these animals exhibit loss of MUC5AC+ mucous cells throughout the airway mucosa of the lung without affecting comparable cells in the tracheal mucosa or MUC5B+ mucous cells in submucosal glands. Similarly, CLCA1-/- pigs exhibit loss of MUC5AC+ mucous cells in the intestinal mucosa without affecting MUC2+ mucous cells. These data establish CLCA1 function for controlling MUC5AC expression as a marker of mucus production and provide a new animal model to study mucus production at respiratory and intestinal sites.

TLR3-Activated Monocyte-Derived Dendritic Cells Trigger Progression from Acute Viral Infection to Chronic Disease in the Lung.

Acute infection is implicated as a trigger for chronic inflammatory disease, but the full basis for this switch is uncertain. In this study, we examine this issue using a mouse model of chronic lung disease that develops after respiratory infection with a natural pathogen (Sendai virus). We investigate this model using a combination of TLR3-deficient mice and adoptive transfer of immune cells into these mice versus the comparable responses in wild-type mice. We found that acute and transient expression of TLR3 on monocyte-derived dendritic cells (moDCs) was selectively required to induce long-term expression of IL-33 and consequent type 2 immune-driven lung disease. Unexpectedly, moDC participation was not based on canonical TLR3 signaling and relied instead on a trophic effect to expand the alveolar epithelial type 2 cell population beyond repair of tissue injury and thereby provide an enriched and persistent cell source of IL-33 required for progression to a disease phenotype that includes lung inflammation, hyperreactivity, excess mucus production, and remodeling. The findings thereby provide a framework wherein viral infection activates TLR3 in moDCs as a front-line immune cell niche upstream of lung epithelial cells to drive the type 2 immune response, leading to chronic inflammatory diseases of the lung (such as asthma and chronic obstructive pulmonary disease in humans) and perhaps progressive and long-term postviral disease in general.

Respiratory Enterovirus (like Parainfluenza Virus) Can Cause Chronic Lung Disease if Protection by Airway Epithelial STAT1 Is Lost.

Epithelial barrier cells are proposed to be critical for host defense, and airway epithelial cell capacity for IFN signal transduction is presumed to protect against respiratory viral infection. However, it has been difficult to fully test these concepts given the absence of tools to analyze IFN signaling specific to airway epithelial cells in vivo. To address these issues, we generated a new line of transgenic mice with Cre-driver genes (Foxj1 and Scgb1a1) for a floxed-Stat1 allele (designated Foxj1-Scgb1a1-Cre-Stat1f/f mice) to target the master IFN signal regulator STAT1 in airway epithelial cells and tested these mice for control of infection because of mouse parainfluenza (Sendai) virus and human enterovirus D68 (EV-D68). Indeed, both types of infections showed increases in viral titers and severity of acute illness in Foxj1-Scgb1a1-Cre-Stat1f/f mice and conventional Stat1-/- mice compared with wild-type mice. In concert, the chronic lung disease that develops after Sendai virus infection was also increased in Foxj1-Scgb1a1-Cre-Stat1f/f and Stat1-/ - mice, marked by airway and adjacent parenchymal immune cell infiltration and mucus production for at least 7 wk postinfection. Unexpectedly, relatively mild EV-D68 infection also progressed to chronic lung disease in Foxj1-Scgb1a1-Cre-Stat1f/f and Stat1 -/- mice but was limited (like viral replication) to airways. The results thereby provide proof-of-concept for a critical role of barrier epithelial cells in protection from acute illness and chronic disease after viral infection and suggest a specific role for airway epithelial cells given the limitation of EV-D68 replication and acute and chronic manifestations of disease primarily to airway tissue.

PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection.

Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve hyper-responsiveness to interferon without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depended on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acted on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to degrade these proteases via the immunoproteasome. Thus, PARP9-DTX3L acted on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway.

Membrane protein Crry maintains homeostasis of the complement system.

Complement activation is tightly regulated to avoid excessive inflammatory and immune responses. Crry(-/-) is an embryonic lethal phenotype secondary to the maternal complement alternative pathway (AP) attacking a placenta deficient in this inhibitor. In this study, we demonstrate that Crry(-/-) mice could be rescued on a partial as well as on a complete factor B (fB)- or C3-deficient maternal background. The C3 and fB protein concentrations in Crry(-/-)C3(+/-) and Crry(-/-)fB(+/-) mice were substantially reduced for gene dosage secondary to enhanced AP turnover. Based on these observations, a breeding strategy featuring reduced maternal AP-activating capacity rescued the lethal phenotype. It led to a novel, stable line of Crry SKO mice carrying normal alleles for C3 and fB. Crry SKO mice also had accelerated C3 and fB turnover and therefore reduced AP- activating potential. These instructive results represent an example of a membrane regulatory protein being responsible for homeostasis of the complement system. They imply that there is constant turnover on cells of the AP pathway which functions as an immune surveillance system for pathogens and altered self.

Phospholipase Cgamma2 modulates integrin signaling in the osteoclast by affecting the localization and activation of Src kinase.

Integrin engagement induces a cascade of signaling pathways that include tyrosine phosphorylation of numerous proteins that lead to modulation of the actin cytoskeleton. Src is a major intracellular mediator of integrin-dependent functions, but the mechanism(s) by which Src is regulated in response to integrin signals is not fully understood. Here, we demonstrate an important role for phospholipase C gamma 2 (PLCgamma2) in Src activation in the osteoclast. Through analysis of primary cells from PLCgamma2(-/-) mice, PLCgamma2 was found to be an important regulator of alpha(v)beta(3) integrin-mediated bone osteoclast cell adhesion, migration, and bone resorption. Adhesion-induced PYK2 and Src phosphorylation is decreased in the absence of PLCgamma2, and the interaction of Src with beta(3) integrin and PYK2 is dramatically reduced. Importantly, PLCgamma2 was found to be required for proper localization of Src to the sealing actin ring, and this function required both its catalytic activity and adapter domains. Based on these results, we propose that PLCgamma2 influences Src activation by mediating the localization of Src to the integrin complex and thereby regulating integrin-mediated functions in the osteoclast.

PLCgamma2 regulates osteoclastogenesis via its interaction with ITAM proteins and GAB2.

Excessive bone loss in arthritic diseases is mostly due to abnormal activation of the immune system leading to stimulation of osteoclasts. While phospholipase Cgamma (PLCgamma) isoforms are known modulators of T and B lymphocyte-mediated immune responses, we found that blockade of PLCgamma enzymatic activity also blocks early osteoclast development and function. Importantly, targeted deletion of Plcg2 in mice led to an osteopetrotic phenotype. PLCgamma2, independent of PLCgamma1, was required for receptor activator of NF-kappaB ligand-induced (RANKL-induced) osteoclastogenesis by differentially regulating nuclear factor of activated T cells c1 (NFATc1), activator protein-1 (AP1), and NF-kappaB. Specifically, we show that NFATc1 upregulation is dependent on RANKL-mediated phosphorylation of PLCgamma2 downstream of Dap12/Fc receptor gamma (Dap12/FcRgamma) receptors and is blocked by the PLCgamma inhibitor U73122. In contrast, activation of JNK and NF-kappaB was not affected by U73122 or Dap12/FcRgamma deletion. Interestingly, we found that in osteoclasts, PLCgamma2 formed a complex with the regulatory adapter molecule GAB2, was required for GAB2 phosphorylation, and modulated GAB2 recruitment to RANK. Thus, PLCgamma2 mediates RANKL-induced osteoclastogenesis and is a potential candidate for antiresorptive therapy.

In vivo correction of complement regulatory protein deficiency with an inhibitor targeting the red blood cell membrane.

Because of the complement system's involvement in many human diseases and potential complications associated with its systemic blockade, site-specific regulation of this effector system is an attractive concept. We report on further developments of such an approach using a single-chain Ab fragment as a vehicle to deliver complement regulatory proteins to a defined cell type. In a model system in which RBCs deficient in complement receptor 1-related gene/protein y (Crry) are rapidly cleared after injection into wild-type animals by a complement-dependent mechanism, we selectively reconstituted these cells with N- and C-terminally targeted recombinant forms of Crry. Transfusion of Crry-coated knockout RBCs into C57BL/6 mice extended their in vivo half-life from <5 min to approximately 2 days. Maintenance of protective levels of Crry (by a combined treatment of donor and recipient RBCs) led to nearly normal RBC survival. Uniform in vitro and in vivo coating of the RBCs and the more efficient complement inhibitory capacity of C-terminally tagged Crry were other interesting features of this experimental system. These results suggest the possibility of using the single-chain Ab fragment-mediated targeting concept of complement regulatory proteins to restrict complement inhibition to the site of its excessive activation.

Negligible role of antibodies and C5 in pregnancy loss associated exclusively with C3-dependent mechanisms through complement alternative pathway.

Factors involved in pregnancy failure due to abnormal fetomaternal tolerance are poorly understood. Here we describe distinct defects in placenta formation and subsequent pregnancy loss solely dependent on the activation of the complement alternative pathway and the effector mechanisms provided by the maternal C3. Surprisingly, this effect is independent of other complement activation pathways and of the effector mechanisms provided by other complement components. These findings provide significant insight into the role of the innate immune system in human pregnancy failure, a frequent clinical outcome.

A role for the Cr2 gene in modifying autoantibody production in systemic lupus erythematosus.

Systemic lupus erythematosus is an autoimmune disease characterized by autoantibody production against nuclear Ags. Recent studies suggest that the Cr2 gene, which encodes for complement receptor (CR)1 and CR2, is important in disease susceptibility. Because the precise disease phenotype related to this gene, in isolation or in relation to other genetic loci, is not known, we analyzed C57BL/6 mice with a targeted mutation in Cr2 (C57BL/6.Cr2(-/-)) with or without a concomitant mutation in Fas (C57BL/6.lpr Cr2(-/-)). The Cr2(null) mutation in a C57BL/6.lpr background markedly increases the serum concentrations of IgG1 and IgG2b and the levels of antinuclear and anti-dsDNA Abs as compared with C57BL/6.lpr controls. There is also a trend for higher concentrations of IgG2a and IgG3. In contrast, isolated deficiencies in either these CRs or Fas have a limited effect in the production of anti-dsDNA Abs. Moreover, the Cr2(null) mutation does not affect other disease manifestations. These findings demonstrate that abnormalities in CR1 and CR2 may be linked to the production of autoantibodies by modifying the effect of other systemic lupus erythematosus susceptibility genes. Phenotypic expression of other disease manifestations need additional Cr2-independent genetic factors.

Complement C3 activation is required for antiphospholipid antibody-induced fetal loss.

The antiphospholipid syndrome (APS) is characterized by recurrent fetal loss, vascular thrombosis, and thrombocytopenia occurring in the presence of antiphospholipid (aPL) antibodies. The pathogenesis of fetal loss and tissue injury in APS is incompletely understood, but is thought to involve platelet and endothelial cell activation as well as procoagulant effects of aPL antibodies acting directly on clotting pathway components. Recent studies have shown that uncontrolled complement activation in the placenta leads to fetal death in utero. We hypothesized that aPL antibodies activate complement in the placenta, generating split products that mediate placental injury and lead to fetal loss and growth retardation. To test this hypothesis, we used a murine model of APS in which pregnant mice are injected with human IgG containing aPL antibodies. We found that inhibition of the complement cascade in vivo, using the C3 convertase inhibitor complement receptor 1-related gene/protein y (Crry)-Ig, blocks fetal loss and growth retardation. Furthermore, mice deficient in complement C3 were resistant to fetal injury induced by aPL antibodies. While antigenic epitopes recognized by aPL antibodies are important in the pathogenesis of APS, our data show that in vivo complement activation is required for aPL antibody-induced fetal loss and growth retardation.