Deciphering the M-cell niche: insights from mouse models on how microfold cells "know" where they are needed.

Known for their distinct antigen-sampling abilities, microfold cells, or M cells, have been well characterized in the gut and other mucosa including the lungs and nasal-associated lymphoid tissue (NALT). More recently, however, they have been identified in tissues where they were not initially suspected to reside, which raises the following question: what external and internal factors dictate differentiation toward this specific role? In this discussion, we will focus on murine studies to determine how these cells are identified (e.g., markers and function) and ask the broader question of factors triggering M-cell localization and patterning. Then, through the consideration of unconventional M cells, which include villous M cells, Type II taste cells, and medullary thymic epithelial M cells (microfold mTECs), we will establish the M cell as not just a player in mucosal immunity but as a versatile niche cell that adapts to its home tissue. To this end, we will consider the lymphoid structure relationship and apical stimuli to better discuss how the differing cellular programming and the physical environment within each tissue yield these cells and their unique organization. Thus, by exploring this constellation of M cells, we hope to better understand the multifaceted nature of this cell in its different anatomical locales.

Route of administration significantly affects particle deposition and cellular recruitment.

Lung exposures to dusts, pollutants, and other aerosol particulates are known to be associated with pulmonary diseases such as asthma and Chronic Obstructive Pulmonary Disease. These health impacts are attributed to the ability of aerosol components to induce pulmonary inflammation, which promotes tissue remodeling, including fibrosis, tissue degradation, and smooth muscle proliferation. Consequently, the distribution of these effects can have a significant impact on the physiologic function of the lung. In order to study the impact of distribution of inhaled particulates on lung pathogenesis, we compared the effect of different methods of particle delivery. By comparing intranasal versus aerosol delivery of fluorescent microspheres, we observed strikingly distinct patterns of particle deposition; intranasal delivery provided focused deposition concentrated on larger airways, while aerosol delivery showed unform deposition throughout the lung parenchyma. Recognizing that the impacts of inflammatory cells are contingent upon their recruitment and behavior, we postulate that these variations in distribution patterns can result in significant alterations in biological responses. To elucidate the relevance of these findings in terms of biological representation, we subsequently conducted an investigation into the responses elicited by the administration of endotoxin (bacterial Lipopolysaccharide, or LPS) in a transgenic neutrophil reporter mouse model. As with the microsphere results, patterns of recruited neutrophil inflammatory responses matched the delivery method; that is, despite the active migratory behavior of neutrophils, inflammatory histopathology patterns were either focused on large airways (intranasal administration) or diffusely throughout the parenchyma (aerosol). These results demonstrate the importance of modes of aerosol delivery as different patterns of inflammation and tissue remodeling will have distinct impacts on lung physiology.

Thymic mimetic cells function beyond self-tolerance.

Development of immunocompetent T cells in the thymus is required for effective defence against all types of pathogens, including viruses, bacteria and fungi. To this end, T cells undergo a very strict educational program in the thymus, during which both non-functional and self-reactive T cell clones are eliminated by means of positive and negative selection1.Thymic epithelial cells (TECs) have an indispensable role in these processes, and previous studies have shown the notable heterogeneity of these cells2-7. Here, using multiomic analysis, we provide further insights into the functional and developmental diversity of TECs in mice, and reveal a detailed atlas of the TEC compartment according to cell transcriptional states and chromatin landscapes. Our analysis highlights unconventional TEC subsets that are similar to functionally well-defined parenchymal populations, including endocrine cells, microfold cells and myocytes. By focusing on the endocrine and microfold TEC populations, we show that endocrine TECs require Insm1 for their development and are crucial to maintaining thymus cellularity in a ghrelin-dependent manner; by contrast, microfold TECs require Spib for their development and are essential for the generation of thymic IgA+ plasma cells. Collectively, our study reveals that medullary TECs have the potential to differentiate into various types of molecularly distinct and functionally defined cells, which not only contribute to the induction of central tolerance, but also regulate the homeostasis of other thymus-resident populations.

Aerosolized aqueous dust extracts collected near a drying lake trigger acute neutrophilic pulmonary inflammation reminiscent of microbial innate immune ligands.

BACKGROUND: A high incidence of asthma is prevalent among residents near the Salton Sea, a large inland terminal lake in southern California. This arid region has high levels of ambient particulate matter (PM); yet while high PM levels are often associated with asthma in many environments, it is possible that the rapidly retreating lake, and exposed playa or lakebed, may contribute components with a specific role in promoting asthma symptoms. OBJECTIVES: Our hypothesis is that asthma may be higher in residents closest to the Salton Sea due to chronic exposures to playa dust. Playa emissions may be concentrating dissolved material from the lake, with microbial components capable of inducing pulmonary innate immune responses. To test this hypothesis, we used a mouse model of aerosol exposures to assess the effects of playa dust. METHODS: From dust collected around the Salton Sea region, aqueous extracts were used to generate aerosols, which were injected into an environmental chamber for mouse exposure studies. We compared the effects of exposure to Salton Sea aerosols, as well as to known immunostimulatory reference materials. Acute 48-h and chronic 7-day exposures were compared, with lungs analyzed for inflammatory cell recruitment and gene expression. RESULTS: Dust from sites nearest to the Salton Sea triggered lung neutrophil inflammation that was stronger at 48-h but reduced at 7-days. This acute inflammatory profile and kinetics resembled the response to innate immune ligands LTA and LPS while distinct from the classic allergic response to Alternaria. CONCLUSION: Lung inflammatory responses to Salton Sea dusts are similar to acute innate immune responses, raising the possibility that microbial components are entrained in the dust, promoting inflammation. This effect highlights the health risks at drying terminal lakes from inflammatory components in dust emissions from exposed lakebed.

Selective Targeting of Tumour Necrosis Factor Receptor 1 Induces Stable Protection from Crohn's-Like Ileitis in TNFΔARE Mice.

BACKGROUND AND AIMS: Crohn's disease is a debilitating chronic inflammatory disorder of the mammalian gastrointestinal tract. Current interventions using anti-tumour necrosis factor [anti-TNF] biologics show long-term benefit in only half of patients. This study focused on the role of the TNF receptor 1 [TNFR1] in pathogenesis in a TNF-driven model of ileitis. METHODS: We studied TNFΔAU-rich element [ARE]/+ [TNFdARE] mice, which develop progressive ileitis similar to Crohn's ileitis. Histopathological analysis and gene expression profiling were used to characterize disease progression from 5 to 16 weeks. Mice with TNFR1 hemizygosity [TNFdARE/R1het] allowed us to assess gene dosage effects. Transcriptional profiling established inflection points in disease progression; inflammatory gene expression increased at 8 weeks with a plateau by 10 weeks, so these were selected as endpoints of treatment using the TNF biologic infliximab and the TNFR1-specific XPro1595. Differences in recruitment of cells in the lamina propria were assessed using flow cytometry. RESULTS: TNFdARE/R1het mice displayed stable long-term protection from disease, associated with decreased recruitment of CD11bhiF4/80lo monocytes and CD11bhiLy6Ghi neutrophils, suggesting an important role of TNFR1 signalling in pathogenesis, and indicating potential benefit from TNFR1-specific intervention. Treatment with infliximab and XPro1595 both showed a similar impact on disease in TNFdARE mice. Importantly, these beneficial effects were greatly surpassed by hemizygosity at the TNFR1 locus. CONCLUSIONS: Treatment with either infliximab or XPro1595 produced moderate protection from ileitis in TNFdARE mice. However, hemizygosity at the TNFR1 locus in TNFdARE mice showed far better protection, implicating TNFR1 signalling as a key mediator of TNF-driven disease.

Salton Sea aerosol exposure in mice induces a pulmonary response distinct from allergic inflammation.

In communities surrounding the Salton Sea, high rates of asthma are associated with high aerosol dust levels. However, the Salton Sea itself may play an additional role in pulmonary health. Therefore, to investigate a potential role of the Salton Sea on pulmonary health, we exposed mice to aerosolized Salton Sea water for 7 days and assessed tissue responses, including cellular infiltration and gene expression changes. For reference, mice were also exposed to aerosolized fungal allergen (Alternaria sp.) and Pacific Ocean aerosols. Exposure to aerosolized Alternaria sp. induced dramatic allergic inflammation, including neutrophil and eosinophil recruitment to the bronchoalveolar lavage fluid (BALF) and lung tissue. By contrast, Salton Sea "spray" induced only B cell recruitment to the lung tissue without increased inflammatory cell numbers in BALF. However, there were consistent gene expression changes suggestive of an inflammatory response. The response to the Salton Sea spray was notably distinct from the response to Pacific Ocean water, which induced some B cell recruitment but without an inflammatory gene expression profile. Our studies suggest that soluble components in Salton Sea water promote induction of a unique inflammation-associated response, though any relationship to asthma remains to be explored.

Novel Mechanical Strain Characterization of Ventilated ex vivo Porcine and Murine Lung using Digital Image Correlation.

Respiratory illnesses, such as bronchitis, emphysema, asthma, and COVID-19, substantially remodel lung tissue, deteriorate function, and culminate in a compromised breathing ability. Yet, the structural mechanics of the lung is significantly understudied. Classical pressure-volume air or saline inflation studies of the lung have attempted to characterize the organ's elasticity and compliance, measuring deviatory responses in diseased states; however, these investigations are exclusively limited to the bulk composite or global response of the entire lung and disregard local expansion and stretch phenomena within the lung lobes, overlooking potentially valuable physiological insights, as particularly related to mechanical ventilation. Here, we present a method to collect the first non-contact, full-field deformation measures of ex vivo porcine and murine lungs and interface with a pressure-volume ventilation system to investigate lung behavior in real time. We share preliminary observations of heterogeneous and anisotropic strain distributions of the parenchymal surface, associative pressure-volume-strain loading dependencies during continuous loading, and consider the influence of inflation rate and maximum volume. This study serves as a crucial basis for future works to comprehensively characterize the regional response of the lung across various species, link local strains to global lung mechanics, examine the effect of breathing frequencies and volumes, investigate deformation gradients and evolutionary behaviors during breathing, and contrast healthy and pathological states. Measurements collected in this framework ultimately aim to inform predictive computational models and enable the effective development of ventilators and early diagnostic strategies.

Intravital Multiphoton Examination of Implant-Associated Staphylococcus aureus Biofilm Infection.

Bacterial infections associated with implanted medical devices represents a healthcare crisis due to their persistence, antibiotic tolerance, and immune avoidance. Indwelling devices are rapidly coated with host plasma and extracellular matrix proteins which can then be exploited by bacterial pathogens for adherence and subsequent biofilm development. Our understanding of the host-pathogen interface that determines the fate of biofilm-mediated infections is limited to the experimental models employed by laboratories studying these organisms. Current in vivo models of biofilm-mediated infection, while certainly useful, are typically limited to end-point analyses of bacterial burden enumeration, immune cell profiling, and cytokine/chemokine analysis. Thus, with these models, the complex, real-time assessment of biofilm development and innate immune cell activity remains imperceptible. Here, we describe a novel murine biofilm infection model employing time-lapse intravital multiphoton microscopy which permits concurrent and real-time visualization of Staphylococcus aureus biofilm formation and immune cell activity. Using cell tracking, we found that S. aureus biofilms impede neutrophil chemotaxis, redirecting their migration patterns to prevent biofilm invasion. This approach is the first to directly examine device-associated biofilm development and host-pathogen interactions and will serve to both further our understanding of infection development and help reveal the effects of future antibiofilm treatment strategies.

Acute Immune Response of Micro- and Nanosized Erythrocyte-Derived Optical Particles in Healthy Mice.

Erythrocyte-derived particles activated by near-infrared (NIR) light present a platform for various phototheranostic applications. We have engineered such a platform with indocyanine green as the NIR-activated agent. A particular feature of these particles is that their diameters can be tuned from micro- to nanoscale, providing a potential capability for broad clinical utility ranging from vascular to cancer-related applications. An important issue related to clinical translation of these particles is their immunogenic effects. Herein, we have evaluated the early-induced innate immune response of these particles in healthy Swiss Webster mice following tail vein injection by measurements of specific cytokines in blood serum, the liver, and the spleen following euthanasia. In particular, we have investigated the effects of particle size and relative dose, time-dependent cytokine response for up to 6 h postinjection, functionalization of the nanosized particles with folate or Herceptin, and dual injections of the particles 1 week apart. Mean concentrations of interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein (MCP)-1 in response to injection of microsized particles at the investigated relative doses were significantly lower than the corresponding mean concentrations induced by lipopolysaccharide (positive control) at 2 h. All investigated doses of the nanosized particles induced significantly higher concentrations of MCP-1 in the liver and the spleen as compared to phosphate buffer saline (PBS) (negative control) at 2 h. In response to micro- and nanosized particles at the highest investigated dose, there were significantly higher levels of TNF-α in blood serum at 2 and 6 h postinjection as compared to the levels associated with PBS treatment at these times. Whereas the mean concentration of TNF-α in the liver significantly increased between 2 and 6 h postinjection in response to the injection of the microsized particles, it was significantly reduced during this time interval in response to the injection of the nanosized particles. In general, functionalization of the nanosized particles was associated with a reduction of IL-6 and MCP-1 in blood serum, the liver, and the spleen, and TNF-α in blood serum. With the exception of IL-10 in the spleen in response to nanosized particles, the second injection of micro- or nanosized particles did not lead to significantly higher concentrations of other cytokines at the investigated dose as compared to a single injection.

Chronic Inflammation in Mucosal Tissues: Barrier Integrity, Inducible Lymphoid Tissues, and Immune Surveillance.

An interesting phenomenon of chronic inflammation is that the associated cytokines can simultaneously promote inflammatory cell recruitment and tissue pathology as well as tissue regeneration and development of inducible organized lymphoid tissues (tertiary lymphoid organs or TLO), demonstrating the remarkable dynamics of the immune interactions with host tissues. In mucosal tissues, chronic immune-mediated inflammation can present a mixed inflammatory pathology including neutrophil infiltrates along with the lymphocytic aggregates. The factors driving this pattern may involve effects on barrier function as well as inducible mechanisms associated with immune surveillance. The relative contribution of these factors may be important in determining the outcome, from resolution to inflammatory stalemate to progressive tissue pathology and destruction. Here, we focus on the specific impact of cytokine-driven inducible lymphoid cells and tissues on immune surveillance at mucosal surfaces, including the induction of epithelial M cells. We propose a model of chronic intestinal inflammation to assess the relative contributions of mucosal barrier integrity, M cell transcytosis of luminal microbes, and inducible lymphoid tissues.

Staphylococcus aureus Fibronectin Binding Protein A Mediates Biofilm Development and Infection.

Implanted medical device-associated infections pose significant health risks, as they are often the result of bacterial biofilm formation. Staphylococcus aureus is a leading cause of biofilm-associated infections which persist due to mechanisms of device surface adhesion, biofilm accumulation, and reprogramming of host innate immune responses. We found that the S. aureus fibronectin binding protein A (FnBPA) is required for normal biofilm development in mammalian serum and that the SaeRS two-component system is required for functional FnBPA activity in serum. Furthermore, serum-developed biofilms deficient in FnBPA were more susceptible to macrophage invasion, and in a model of biofilm-associated implant infection, we found that FnBPA is crucial for the establishment of infection. Together, these findings show that S. aureus FnBPA plays an important role in physical biofilm development and represents a potential therapeutic target for the prevention and treatment of device-associated infections.

A randomized trial of a lab-embedded discourse intervention to improve research ethics.

We report a randomized trial of a research ethics training intervention designed to enhance ethics communication in university science and engineering laboratories, focusing specifically on authorship and data management. The intervention is a project-based research ethics curriculum that was designed to enhance the ability of science and engineering research laboratory members to engage in reason giving and interpersonal communication necessary for ethical practice. The randomized trial was fielded in active faculty-led laboratories at two US research-intensive institutions. Here, we show that laboratory members perceived improvements in the quality of discourse on research ethics within their laboratories and enhanced awareness of the relevance and reasons for that discourse for their work as measured by a survey administered over 4 mo after the intervention. This training represents a paradigm shift compared with more typical module-based or classroom ethics instruction that is divorced from the everyday workflow and practices within laboratories and is designed to cultivate a campus culture of ethical science and engineering research in the very work settings where laboratory members interact.

Crosslinked flagella as a stabilized vaccine adjuvant scaffold.

BACKGROUND: Engineered vaccine proteins incorporating both antigen and adjuvant components are constructed with the aim of combining functions to induce effective protective immunity. Bacterial flagellin is a strong candidate for an engineered vaccine scaffold as it is known to provide adjuvant activity through its TLR5 and inflammasome activation. Moreover, polymerized flagellin filaments can elicit a more robust immunoglobulin response than monomeric flagellin, and the multimeric antigen form can also promote T cell-independent antibody responses. Here, we aim to produce and test a covalently stabilized polymerized flagellar filament, providing additional immune efficacy through stabilization of its polymeric filament structure, as well as stabilization for long-term storage. RESULTS: Computational modeling of monomer packing in flagellin filaments helped identify amino acids with proximity to neighboring flagella protofilaments. Paired cysteine substitutions were made at amino acids predicted to form inter-monomer disulfide cross-links, and these substitutions were capable of forming flagella when transfected into a flagellin-negative strain of Salmonella enterica subspecies Typhimurium. Interestingly, each paired substitution stabilized different helical conformational polymorphisms; the stabilized filaments lost the ability to transition between conformations, reducing bacterial motility. More importantly, the paired substitutions enabled extensive disulfide cross links and intra-filament multimer formation, and in one of the three variants, permitted filament stability in high acidic and temperature conditions where wild-type filaments would normally rapidly depolymerize. In addition, with regard to potential adjuvant activity, all crosslinked flagella filaments were able to induce wild-type levels of epithelial NF-κB in a cell reporter system. Finally, bacterial virulence was unimpaired in epithelial adherence and invasion, and the cysteine substitutions also appeared to increase bacterial resistance to oxidizing and reducing conditions. CONCLUSIONS: We identified amino acid pairs, with cysteine substitutions, were able to form intermolecular disulfide bonds that stabilized the resulting flagellar filaments in detergent, hydrochloric acid, and high temperatures while retaining its immunostimulatory function. Flagellar filaments with disulfide-stabilized protofilaments introduce new possibilities for the application of flagella as a vaccine adjuvant. Specifically, increased stability and heat tolerance permits long-term storage in a range of temperature environments, as well as delivery under a range of clinical conditions.

M Cells: Intelligent Engineering of Mucosal Immune Surveillance.

M cells are specialized intestinal epithelial cells that provide the main machinery for sampling luminal microbes for mucosal immune surveillance. M cells are usually found in the epithelium overlying organized mucosal lymphoid tissues, but studies have identified multiple distinct lineages of M cells that are produced under different conditions, including intestinal inflammation. Among these lineages there is a common morphology that helps explain the efficiency of M cells in capturing luminal bacteria and viruses; in addition, M cells recruit novel cellular mechanisms to transport the particles across the mucosal barrier into the lamina propria, a process known as transcytosis. These specializations used by M cells point to a novel engineering of cellular machinery to selectively capture and transport microbial particles of interest. Because of the ability of M cells to effectively violate the mucosal barrier, the circumstances of M cell induction have important consequences. Normal immune surveillance insures that transcytosed bacteria are captured by underlying myeloid/dendritic cells; in contrast, inflammation can induce development of new M cells not accompanied by organized lymphoid tissues, resulting in bacterial transcytosis with the potential to amplify inflammatory disease. In this review, we will discuss our own perspectives on the life history of M cells and also raise a few questions regarding unique aspects of their biology among epithelia.

Establishment and characterization of a multi-purpose large animal exposure chamber for investigating health effects.

Air pollution poses a significant threat to the environment and human health. Most in vivo health studies conducted regarding air pollutants, including particulate matter (PM) and gas phase pollutants, have been either through traditional medical intranasal treatment or using a tiny chamber, which limit animal activities. In this study, we designed and tested a large, whole-body, multiple animal exposure chamber with uniform dispersion and exposure stability for animal studies. The chamber simultaneously controls particle size distribution and PM mass concentration. Two different methods were used to generate aerosol suspension through either soluble material (Alternaria extract), liquid particle suspension (nanosilica solution), or dry powder (silica powder). We demonstrate that the chamber system provides well controlled and characterized whole animal exposures, where dosage is by inhalation of particulate matter.

Hematopoietic cell-derived RELMα regulates hookworm immunity through effects on macrophages.

Resistin-like molecule α (RELMα) is a highly secreted protein in type 2 (Th2) cytokine-induced inflammation including helminth infection and allergy. In infection with Nippostrongylus brasiliensis (Nb), RELMα dampens Th2 inflammatory responses. RELMα is expressed by immune cells, and by epithelial cells (EC); however, the functional impact of immune versus EC-derived RELMα is unknown. We generated bone marrow (BM) chimeras that were RELMα deficient (RELMα-/- ) in BM or non BM cells and infected them with Nb. Non BM RELMα-/- chimeras had comparable inflammatory responses and parasite burdens to RELMα+/+ mice. In contrast, both RELMα-/- and BM RELMα-/- mice exhibited increased Nb-induced lung and intestinal inflammation, correlated with elevated Th2 cytokines and Nb killing. CD11c+ lung macrophages were the dominant BM-derived source of RELMα and can mediate Nb killing. Therefore, we employed a macrophage-worm co-culture system to investigate whether RELMα regulates macrophage-mediated Nb killing. Compared to RELMα+/+ macrophages, RELMα-/- macrophages exhibited increased binding to Nb and functionally impaired Nb development. Supplementation with recombinant RELMα partially reversed this phenotype. Gene expression analysis revealed that RELMα decreased cell adhesion and Fc receptor signaling pathways, which are associated with macrophage-mediated helminth killing. Collectively, these studies demonstrate that BM-derived RELMα is necessary and sufficient to dampen Nb immune responses, and identify that one mechanism of action of RELMα is through inhibiting macrophage recruitment and interaction with Nb. Our findings suggest that RELMα acts as an immune brake that provides mutually beneficial effects for the host and parasite by limiting tissue damage and delaying parasite expulsion.

Continuous Inhalation Exposure to Fungal Allergen Particulates Induces Lung Inflammation While Reducing Innate Immune Molecule Expression in the Brainstem.

Continuous exposure to aerosolized fine (particle size ≤2.5 µm) and ultrafine (particle size ≤0.1 µm) particulates can trigger innate inflammatory responses in the lung and brain depending on particle composition. Most studies of manmade toxicants use inhalation exposure routes, whereas most studies of allergens use soluble solutions administered via intranasal or injection routes. Here, we tested whether continuous inhalation exposure to aerosolized Alternaria alternata particulates (a common fungal allergen associated with asthma) would induce innate inflammatory responses in the lung and brain. By designing a new environmental chamber able to control particle size distribution and mass concentration, we continuously exposed adult mice to aerosolized ultrafine Alternaria particulates for 96 hr. Despite induction of innate immune responses in the lung, induction of innate immune responses in whole brain samples was not detected by quantitative polymerase chain reaction or flow cytometry. However, exposure did trigger decreases in Arginase 1, inducible nitric oxide synthase, and tumor necrosis factor alpha mRNA in the brainstem samples containing the central nervous system respiratory circuit (the dorsal respiratory group, ventral respiratory group, and the pre-Bötzinger and Bötzinger complexes). In addition, a significant decrease in the percentage of Toll-like receptor 2-expressing brainstem microglia was detected by flow cytometry. Histologic analysis revealed a significant decrease in Iba1 but not glial fibrillary acidic protein immunoreactivity in both the brainstem and the hippocampus. Together these data indicate that inhalation exposure to a natural fungal allergen under conditions sufficient to induce lung inflammation surprisingly causes reductions in baseline expression of select innate immune molecules (similar to that observed during endotoxin tolerance) in the region of the central nervous system controlling respiration.

Vigilance or Subversion? Constitutive and Inducible M Cells in Mucosal Tissues.

Microfold (M) cells are epithelial cells present in mucosal tissues and specialized for the capture of luminal microparticles and their delivery to underlying immune cells; thus, they are crucial participants in mucosal immune surveillance. Multiple phenotypic subsets of M cells have now been described, all sharing a unique apical morphology that provides clues to their ability to capture microbial particles. The existence of diverse M cell phenotypes, especially inflammation-inducible M cells, provides an intriguing puzzle: some variants may augment luminal surveillance to boost mucosal immunity, while others may promote microbial access to tissues. Here, I consider the unique induction requirements of each M cell subset and functional differences, highlighting the potentially distinct consequences in mucosal immunity.

Protein-Nanoparticle Hydrogels That Self-assemble in Response to Peptide-Based Molecular Recognition.

Recently, supramolecular hydrogels assembled through nonspecific interactions between polymers and nanoparticles (termed PNP systems) were reported to have rapid shear-thinning and self-healing properties amenable for cell-delivery applications in regenerative medicine. Here, we introduce protein engineering concepts into the design of a new family of PNP hydrogels to enable direct control over the polymer-nanoparticle interactions using peptide-based molecular recognition motifs. Specifically, we have designed a bifunctional peptide that induces supramolecular hydrogel assembly between hydroxy apatite nanoparticles and an engineered, recombinant protein. We demonstrate that this supramolecular assembly critically requires molecular recognition, as no assembly is observed in the presence of control peptides with a scrambled amino acid sequence. Titration of the bifunctional peptide enables direct control over the number of physical cross-links within the system and hence the resulting hydrogel mechanical properties. As with previous PNP systems, these materials are rapidly shear-thinning and self-healing. As proof-of-concept, we demonstrate that these materials are suitable for therapeutic cell delivery applications in a preclinical murine calvarial defect model.