The lung environment controls alveolar macrophage metabolism and responsiveness in type 2 inflammation.

Fine control of macrophage activation is needed to prevent inflammatory disease, particularly at barrier sites such as the lungs. However, the dominant mechanisms that regulate the activation of pulmonary macrophages during inflammation are poorly understood. We found that alveolar macrophages (AlvMs) were much less able to respond to the canonical type 2 cytokine IL-4, which underpins allergic disease and parasitic worm infections, than macrophages from lung tissue or the peritoneal cavity. We found that the hyporesponsiveness of AlvMs to IL-4 depended upon the lung environment but was independent of the host microbiota or the lung extracellular matrix components surfactant protein D (SP-D) and mucin 5b (Muc5b). AlvMs showed severely dysregulated metabolism relative to that of cavity macrophages. After removal from the lungs, AlvMs regained responsiveness to IL-4 in a glycolysis-dependent manner. Thus, impaired glycolysis in the pulmonary niche regulates AlvM responsiveness during type 2 inflammation.

Chitinase-like proteins promote IL-17-mediated neutrophilia in a tradeoff between nematode killing and host damage.

Enzymatically inactive chitinase-like proteins (CLPs) such as BRP-39, Ym1 and Ym2 are established markers of immune activation and pathology, yet their functions are essentially unknown. We found that Ym1 and Ym2 induced the accumulation of neutrophils through the expansion of γδ T cell populations that produced interleukin 17 (IL-17). While BRP-39 did not influence neutrophilia, it was required for IL-17 production in γδ T cells, which suggested that regulation of IL-17 is an inherent feature of mouse CLPs. Analysis of a nematode infection model, in which the parasite migrates through the lungs, revealed that the IL-17 and neutrophilic inflammation induced by Ym1 limited parasite survival but at the cost of enhanced lung injury. Our studies describe effector functions of CLPs consistent with innate host defense traits of the chitinase family.

Interleukin-4 Receptor α Signaling in Myeloid Cells Controls Collagen Fibril Assembly in Skin Repair.

Activation of the immune response during injury is a critical early event that determines whether the outcome of tissue restoration is regeneration or replacement of the damaged tissue with a scar. The mechanisms by which immune signals control these fundamentally different regenerative pathways are largely unknown. We have demonstrated that, during skin repair in mice, interleukin-4 receptor α (IL-4Rα)-dependent macrophage activation controlled collagen fibril assembly and that this process was important for effective repair while having adverse pro-fibrotic effects. We identified Relm-α as one important player in the pathway from IL-4Rα signaling in macrophages to the induction of lysyl hydroxylase 2 (LH2), an enzyme that directs persistent pro-fibrotic collagen cross-links, in fibroblasts. Notably, Relm-ß induced LH2 in human fibroblasts, and expression of both factors was increased in lipodermatosclerosis, a condition of excessive human skin fibrosis. Collectively, our findings provide mechanistic insights into the link between type 2 immunity and initiation of pro-fibrotic pathways.

The magnitude of airway remodeling is not altered by distinct allergic inflammatory responses in BALB/c versus C57BL/6 mice but matrix composition differs.

Allergic airway inflammation is heterogeneous with variability in immune phenotypes observed across asthmatic patients. Inflammation has been thought to directly contribute to airway remodeling in asthma, but clinical data suggest that neutralizing type 2 cytokines does not necessarily alter disease pathogenesis. Here, we utilized C57BL/6 and BALB/c mice to investigate the development of allergic airway inflammation and remodeling. Exposure to an allergen cocktail for up to 8 weeks led to type 2 and type 17 inflammation, characterized by airway eosinophilia and neutrophilia and increased expression of chitinase-like proteins in both C57BL/6 and BALB/c mice. However, BALB/c mice developed much greater inflammatory responses than C57BL/6 mice, effects possibly explained by a failure to induce pathways that regulate and maintain T-cell activation in C57BL/6 mice, as shown by whole lung RNA transcript analysis. Allergen administration resulted in a similar degree of airway remodeling between mouse strains but with differences in collagen subtype composition. Increased collagen III was observed around the airways of C57BL/6 but not BALB/c mice while allergen-induced loss of basement membrane collagen IV was only observed in BALB/c mice. This study highlights a model of type 2/type 17 airway inflammation in mice whereby development of airway remodeling can occur in both BALB/c and C57BL/6 mice despite differences in immune response dynamics between strains. Importantly, compositional changes in the extracellular matrix between genetic strains of mice may help us better understand the relationships between lung function, remodeling and airway inflammation.

Inflammasome-Independent Role for NLRP3 in Controlling Innate Antihelminth Immunity and Tissue Repair in the Lung.

Alternatively activated macrophages are essential effector cells during type 2 immunity and tissue repair following helminth infections. We previously showed that Ym1, an alternative activation marker, can drive innate IL-1R-dependent neutrophil recruitment during infection with the lung-migrating nematode, Nippostrongylus brasiliensis, suggesting a potential role for the inflammasome in the IL-1-mediated innate response to infection. Although inflammasome proteins such as NLRP3 have important proinflammatory functions in macrophages, their role during type 2 responses and repair are less defined. We therefore infected Nlrp3 -/- mice with N. brasiliensis Unexpectedly, compared with wild-type (WT) mice, infected Nlrp3 -/- mice had increased neutrophilia and eosinophilia, correlating with enhanced worm killing but at the expense of increased tissue damage and delayed lung repair. Transcriptional profiling showed that infected Nlrp3 -/- mice exhibited elevated type 2 gene expression compared with WT mice. Notably, inflammasome activation was not evident early postinfection with N. brasiliensis, and in contrast to Nlrp3 -/- mice, antihelminth responses were unaffected in caspase-1/11-deficient or WT mice treated with the NLRP3-specific inhibitor MCC950. Together these data suggest that NLRP3 has a role in constraining lung neutrophilia, helminth killing, and type 2 immune responses in an inflammasome-independent manner.

The Requirement of Genetic Diagnostic Technologies for Environmental Surveillance of Antimicrobial Resistance.

Antimicrobial resistance (AMR) is threatening modern medicine. While the primary cost of AMR is paid in the healthcare domain, the agricultural and environmental domains are also reservoirs of resistant microorganisms and hence perpetual sources of AMR infections in humans. Consequently, the World Health Organisation and other international agencies are calling for surveillance of AMR in all three domains to guide intervention and risk reduction strategies. Technologies for detecting AMR that have been developed for healthcare settings are not immediately transferable to environmental and agricultural settings, and limited dialogue between the domains has hampered opportunities for cross-fertilisation to develop modified or new technologies. In this feature, we discuss the limitations of currently available AMR sensing technologies used in the clinic for sensing in other environments, and what is required to overcome these limitations.

Ym1 induces RELMα and rescues IL-4Rα deficiency in lung repair during nematode infection.

Ym1 and RELMα are established effector molecules closely synonymous with Th2-type inflammation and associated pathology. Here, we show that whilst largely dependent on IL-4Rα signaling during a type 2 response, Ym1 and RELMα also have IL-4Rα-independent expression patterns in the lung. Notably, we found that Ym1 has opposing effects on type 2 immunity during nematode infection depending on whether it is expressed at the time of innate or adaptive responses. During the lung migratory stage of Nippostrongylus brasiliensis, Ym1 promoted the subsequent reparative type 2 response but once that response was established, IL-4Rα-dependent Ym1 was important for limiting the magnitude of type 2 cytokine production from both CD4+ T cells and innate lymphoid cells in the lung. Importantly, our study demonstrates that delivery of Ym1 to IL-4Rα deficient animals drives RELMα production and overcomes lung repair deficits in mice deficient in type 2 immunity. Together, Ym1 and RELMα, exhibit time and dose-dependent interactions that determines the outcome of lung repair during nematode infection.

Macrophage origin limits functional plasticity in helminth-bacterial co-infection.

Rapid reprogramming of the macrophage activation phenotype is considered important in the defense against consecutive infection with diverse infectious agents. However, in the setting of persistent, chronic infection the functional importance of macrophage-intrinsic adaptation to changing environments vs. recruitment of new macrophages remains unclear. Here we show that resident peritoneal macrophages expanded by infection with the nematode Heligmosomoides polygyrus bakeri altered their activation phenotype in response to infection with Salmonella enterica ser. Typhimurium in vitro and in vivo. The nematode-expanded resident F4/80high macrophages efficiently upregulated bacterial induced effector molecules (e.g. MHC-II, NOS2) similarly to newly recruited monocyte-derived macrophages. Nonetheless, recruitment of blood monocyte-derived macrophages to Salmonella infection occurred with equal magnitude in co-infected animals and caused displacement of the nematode-expanded, tissue resident-derived macrophages from the peritoneal cavity. Global gene expression analysis revealed that although nematode-expanded resident F4/80high macrophages made an anti-bacterial response, this was muted as compared to newly recruited F4/80low macrophages. However, the F4/80high macrophages adopted unique functional characteristics that included enhanced neutrophil-stimulating chemokine production. Thus, our data provide important evidence that plastic adaptation of MΦ activation does occur in vivo, but that cellular plasticity is outweighed by functional capabilities specific to the tissue origin of the cell.

Rational design of new materials using recombinant structural proteins: Current state and future challenges.

Sequence-definable polymers are seen as a prerequisite for design of future materials, with many polymer scientists regarding such polymers as the holy grail of polymer science. Recombinant proteins are sequence-defined polymers. Proteins are dictated by DNA templates and therefore the sequence of amino acids in a protein is defined, and molecular biology provides tools that allow redesign of the DNA as required. Despite this advantage, proteins are underrepresented in materials science. In this publication we investigate the advantages and limitations of using proteins as templates for rational design of new materials.

Chitinase-like proteins as regulators of innate immunity and tissue repair: helpful lessons for asthma?

Chitinases and chitinase-like proteins (CLPs) belong to the glycoside hydrolase family 18 of proteins. Chitinases are expressed in mammals and lower organisms, facilitate chitin degradation, and hence act as host-defence enzymes. Gene duplication and loss-of-function mutations of enzymatically active chitinases have resulted in the expression of a diverse range of CLPs across different species. CLPs are genes that are increasingly associated with inflammation and tissue remodelling not only in mammals but also across distant species. While the focus has remained on understanding the functions and expression patterns of CLPs during disease in humans, studies in mouse and lower organisms have revealed important and overlapping roles of the CLP family during physiology, host defence and pathology. This review will summarise recent insights into the regulatory functions of CLPs on innate immune pathways and discuss how these effects are not only important for host defence and tissue injury/repair after pathogen invasion, but also how they have extensive implications for pathological processes involved in diseases such as asthma.

Host protective roles of type 2 immunity: parasite killing and tissue repair, flip sides of the same coin.

Metazoan parasites typically induce a type 2 immune response, characterized by T helper 2 (Th2) cells that produce the cytokines IL-4, IL-5 and IL-13 among others. The type 2 response is host protective, reducing the number of parasites either through direct killing in the tissues, or expulsion from the intestine. Type 2 immunity also protects the host against damage mediated by these large extracellular parasites as they migrate through the body. At the center of both the innate and adaptive type 2 immune response, is the IL-4Rα that mediates many of the key effector functions. Here we highlight the striking overlap between the molecules, cells and pathways that mediate both parasite control and tissue repair. We have proposed that adaptive Th2 immunity evolved out of our innate repair pathways to mediate both accelerated repair and parasite control in the face of continual assault from multicellular pathogens. Type 2 cytokines are involved in many aspects of mammalian physiology independent of helminth infection. Therefore understanding the evolutionary relationship between helminth killing and tissue repair should provide new insight into immune mechanisms of tissue protection in the face of physical injury.

Recombinant Structural Proteins and Their Use in Future Materials.

Recombinant proteins are polymers that offer the materials engineer absolute control over chain length and composition: key attributes required for design of advanced polymeric materials. Through this control, these polymers can be encoded to contain information that enables them to respond as the environment changes. However, despite their promise, protein-based materials are under-represented in materials science. In this chapter we investigate why this is and describe recent efforts to address this. We discuss constraints limiting rational design of structural proteins for advanced materials; advantages and disadvantages of different recombinant expression platforms; and, methods to fabricate proteins into solid-state materials. Finally, we describe the silk proteins used in our laboratory as templates for information-containing polymers.

IL-33 delivery induces serous cavity macrophage proliferation independent of interleukin-4 receptor alpha.

IL-33 plays an important role in the initiation of type-2 immune responses, as well as the enhancement of type 2 effector functions. Engagement of the IL-33 receptor on macrophages facilitates polarization to an alternative activation state by amplifying IL-4 and IL-13 signaling to IL-4Rα. IL-4 and IL-13 also induce macrophage proliferation but IL-33 involvement in this process has not been rigorously evaluated. As expected, in vivo delivery of IL-33 induced IL-4Rα-dependent alternative macrophage activation in the serous cavities. IL-33 delivery also induced macrophages to proliferate but, unexpectedly, this was independent of IL-4Rα signaling. In a filarial nematode infection model in which IL-4Rα-dependent alternative activation and proliferation in the pleural cavity is well described, IL-33R was essential for alternative activation but not macrophage proliferation. Similarly, during Alternaria alternata induced airway inflammation, which provokes strong IL-33 responses, we observed that both IL-4Rα and IL-33R were required for alternative activation, while macrophage proliferation in the pleural cavity was still evident in the absence of either receptor alone. Our data show that IL-33R and IL-4Rα promote macrophage proliferation independently of each other, but both are essential for induction of alternative activation.

Structural Analysis of Hand Drawn Bumblebee Bombus terrestris Silk.

Bombus terrestris, commonly known as the buff-tailed bumblebee, is native to Europe, parts of Africa and Asia. It is commercially bred for use as a pollinator of greenhouse crops. Larvae pupate within a silken cocoon that they construct from proteins produced in modified salivary glands. The amino acid composition and protein structure of hand drawn B. terrestris, silk fibres was investigated through the use of micro-Raman spectroscopy. Spectra were obtained from single fibres drawn from the larvae salivary gland at a rate of 0.14 cm/s. Raman spectroscopy enabled the identification of poly(alanine), poly(alanine-glycine), phenylalanine, tryptophan, and methionine, which is consistent with the results of amino acid analysis. The dominant protein conformation was found to be coiled coil (73%) while the ß-sheet content of 10% is, as expected, lower than those reported for hornets and ants. Polarized Raman spectra revealed that the coiled coils were highly aligned along the fibre axis while the ß-sheet and random coil components had their peptide carbonyl groups roughly perpendicular to the fibre axis. The protein orientation distribution is compared to those of other natural and recombinant silks. A structural model for the B. terrestris silk fibre is proposed based on these results.

More than one way to spin a crystallite: multiple trajectories through liquid crystallinity to solid silk.

Arthropods face several key challenges in processing concentrated feedstocks of proteins (silk dope) into solid, semi-crystalline silk fibres. Strikingly, independently evolved lineages of silk-producing organisms have converged on the use of liquid crystal intermediates (mesophases) to reduce the viscosity of silk dope and assist the formation of supramolecular structure. However, the exact nature of the liquid-crystal-forming-units (mesogens) in silk dope, and the relationship between liquid crystallinity, protein structure and silk processing is yet to be fully elucidated. In this review, we focus on emerging differences in this area between the canonical silks containing extended-ß-sheets made by silkworms and spiders, and 'non-canonical' silks made by other insect taxa in which the final crystallites are coiled-coils, collagen helices or cross-ß-sheets. We compared the amino acid sequences and processing of natural, regenerated and recombinant silk proteins, finding that canonical and non-canonical silk proteins show marked differences in length, architecture, amino acid content and protein folding. Canonical silk proteins are long, flexible in solution and amphipathic; these features allow them both to form large, micelle-like mesogens in solution, and to transition to a crystallite-containing form due to mechanical deformation near the liquid-solid transition. By contrast, non-canonical silk proteins are short and have rod or lath-like structures that are well suited to act both as mesogens and as crystallites without a major intervening phase transition. Given many non-canonical silk proteins can be produced at high yield in E. coli, and that mesophase formation is a versatile way to direct numerous kinds of supramolecular structure, further elucidation of the natural processing of non-canonical silk proteins may to lead to new developments in the production of advanced protein materials.

Convergently-evolved structural anomalies in the coiled coil domains of insect silk proteins.

The use of coiled coil proteins as the basis of silk materials is an engineering solution that has evolved convergently in at least five insect lineages-the stinging hymenopterans (ants, bees, hornets), argid sawflies, fleas, lacewings, and praying mantises-and persisted throughout large radiations of these insect families. These coiled coil silk proteins share a characteristic distinct from other coiled coil proteins, in that they are fabricated into solid materials after accumulating as highly concentrated solutions within dedicated glands. Here, we relate the amino acid sequences of these proteins to the secondary and tertiary structural information available from biophysical methods such as X-ray scattering, nuclear magnetic resonance and Raman spectroscopy. We investigate conserved and convergently evolved features within these proteins and compare these to the features of classic coiled coil proteins including tropomyosin and leucine zippers. Our analysis finds that the coiled coil domains of insect silk proteins have several common structural anomalies including a high prevalence of alanine residues in core positions. These atypical features of the coiled coil fibrous proteins - which likely produce deviations from canonical coiled-coil structure - likely exist due to selection pressures related to the process of silk fabrication and the final function of the proteins.

Induction of IL-4Rα-dependent microRNAs identifies PI3K/Akt signaling as essential for IL-4-driven murine macrophage proliferation in vivo.

Macrophage (MΦ) activation must be tightly controlled to preclude overzealous responses that cause self-damage. MicroRNAs promote classical MΦ activation by blocking antiinflammatory signals and transcription factors but also can prevent excessive TLR signaling. In contrast, the microRNA profile associated with alternatively activated MΦ and their role in regulating wound healing or antihelminthic responses has not been described. By using an in vivo model of alternative activation in which adult Brugia malayi nematodes are implanted surgically in the peritoneal cavity of mice, we identified differential expression of miR-125b-5p, miR-146a-5p, miR-199b-5p, and miR-378-3p in helminth-induced MΦ. In vitro experiments demonstrated that miR-378-3p was specifically induced by IL-4 and revealed the IL-4-receptor/PI3K/Akt-signaling pathway as a target. Chemical inhibition of this pathway showed that intact Akt signaling is an important enhancement factor for alternative activation in vitro and in vivo and is essential for IL-4-driven MΦ proliferation in vivo. Thus, identification of miR-378-3p as an IL-4Rα-induced microRNA led to the discovery that Akt regulates the newly discovered mechanism of IL-4-driven macrophage proliferation. Together, the data suggest that negative regulation of Akt signaling via microRNAs might play a central role in limiting MΦ expansion and alternative activation during type 2 inflammatory settings.

A comparison of convergently evolved insect silks that share ß-sheet molecular structure.

Raspy crickets produce silk webs that are used to build shelters. These webs have been found to consist of both fiber and film components. Raman spectra obtained from both components were found to be very similar for a given species. The protein structure of the fibers and films produced by both species was predominately ß-sheet with lesser amounts of ß-turns, unordered and α-helical protein also detected. The orientation of the ß-sheet backbone in the fiber was determined to be parallel to the fiber axis. Compared to cocoon and dragline silk the orientation distribution exhibits a significant randomly orientated protein component. Amino acid analysis confirmed the presence of glycine, serine, and alanine in both species, which are known to form antiparallel ß-sheet structures. Both species, although at significantly different concentrations, where found to contain proline. This amino acid is uncommon in insect silks, and likely involved in increasing fiber elasticity.