Mucosal Sites Research Articles

T4 bacteriophage nanoparticles engineered through CRISPR provide a versatile platform for rapid development of flu mucosal vaccines

Vaccines that trigger mucosal immune responses at the entry portals of pathogens are highly desired. Here, we showed that antigen-decorated nanoparticle generated through CRISPR engineering of T4 bacteriophage can serve as a universal platform for the rapid development of mucosal vaccines. Insertion of Flu viral M2e into phage T4 genome through fusion to Soc (Small Outer Capsid protein) generated a recombinant phage, and the Soc-M2e proteins self-assembled onto phage capsids to form 3M2e-T4 nanoparticles during propagation of T4 in E. coli. Intranasal administration of 3M2e-T4 nanoparticles maintains antigen persistence in the lungs, resulting in increased uptake and presentation by antigen-presenting cells. M2e-specific secretory IgA, effector (TEM), central (TCM), and tissue-resident memory CD4+ T cells (TRM) were efficiently induced in the local mucosal sites, which mediated protections against divergent influenza viruses. Our studies demonstrated the mechanisms of immune protection following 3M2e-T4 nanoparticles vaccination and provide a versatile T4 platform that can be customized to rapidly develop mucosal vaccines against future emerging epidemics.

Influenza vaccine format mediates distinct cellular and antibody responses in human immune organoids

Highly effective vaccines elicit specific, robust, and durable adaptive immune responses. To advance informed vaccine design, it is critical that we understand the cellular dynamics underlying responses to different antigen formats. Here, we sought to understand how antigen-specific B and Tcells were activated and participated in adaptive immune responses within the mucosal site. Using a human tonsil organoid model, we tracked the differentiation and kinetics of the adaptive immune response to influenza vaccine and virus modalities. Each antigen format elicited distinct B and Tcell responses, including differences in their magnitude, diversity, phenotype, function, and breadth. These differences culminated in substantial changes in the corresponding antibody response. A major source of antigen format-related variability was the ability to recruit naive vs. memory B and Tcells to the response. These findings have important implications for vaccine design and the generation of protective immune responses in the upper respiratory tract.

IKK2/NFkB signaling controls lung resident CD8+ T cell memory during influenza infection

CD8+ T cell tissue resident memory (TRM) cells are especially suited to control pathogen spread at mucosal sites. However, their maintenance in lung is short-lived. TCR-dependent NFkB signaling is crucial for T cell memory but how and when NFkB signaling modulates tissue resident and circulating T cell memory during the immune response is unknown. Here, we find that enhancing NFkB signaling in T cells once memory to influenza is established, increases pro-survival Bcl-2 and CD122 levels thus boosting lung CD8+ TRM maintenance. By contrast, enhancing NFkB signals during the contraction phase of the response leads to a defect in CD8+ TRM differentiation without impairing recirculating memory subsets. Specifically, inducible activation of NFkB via constitutive active IKK2 or TNF interferes with TGFβ signaling, resulting in defects of lung CD8+ TRM imprinting molecules CD69, CD103, Runx3 and Eomes. Conversely, inhibiting NFkB signals not only recovers but improves the transcriptional signature and generation of lung CD8+ TRM. Thus, NFkB signaling is a critical regulator of tissue resident memory, whose levels can be tuned at specific times during infection to boost lung CD8+ TRM.

The Common Mucosal System Fifty Years on: From Cell Traffic in the Rabbit to Immune Resilience to SARS-CoV-2 Infection by Shifting Risk within Normal and Disease Populations.

The idea of a common mucosal immune system (CMS) is 50 years old. Its relevance to immune protection at mucosal sites and its potential to modulate the impact of vaccination-induced protection against infection of the airway has been poorly understood. The consequent failure of the current SARS-CoV-2 vaccination to satisfy expectations with respect to prevention of infection, viral transmission, duration of protection, and pattern of clinical protection, led to public health and medical decisions now under review. This review summarises knowledge of the CMS in man, including the powerful role it plays in immune protection and lessons with respect to what can and cannot be achieved by systemic and mucosal vaccination for the prevention of airway infection. The powerful impact in both health and disease of optimising delivery of immune protection using selected isolates from the respiratory microbiome is demonstrated through a review of randomised controlled trials (RCTs) in subjects with chronic airway disease, and in otherwise healthy individuals with risk factors, in whom the idea of mucosal immune resilience is introduced. This review is dedicated to two giants of mucosal immunology: Professors John Bienenstock and Allan Cripps. Their recent deaths are keenly felt by their colleagues and students.

Two Head and Neck Carcinomas With Squamous and Mucinous Components and Human Papillomavirus Associations: Maxillary Mucoepidermoid Carcinoma ex Sinonasal Schneiderian Papilloma and Tonsillar Invasive Stratified Mucin Producing Carcinoma (ISMC).

Carcinomas of the head-and-neck region with squamous and glandular/mucinous features constitute a heterogeneous group, with a significant minority of tumors showing an human papillomavirus (HPV) association. The differential diagnosis is usually between mucoepidermoid carcinoma (MEC) and adenosquamous carcinoma. We present here two tumors that exemplify both the challenges of diagnostic classification, as well as the complex relationship to HPV: (a) a low risk HPV positive/p16 negative carcinoma that is most consistent with a relatively typical intermediate grade mucoepidermoid type carcinoma with complete MEC phenotype (three cell types), originating from intranasal sinonasal papillomas with exophytic and inverted patterns, and invading surrounding maxillary compartments, and (b) a p16 and keratin 7 (KRT7) positive carcinoma of the right tonsil, characterized by stratified squamous and mucinous cell (mucocyte) features. Whereas the first tumor represents a typical MEC ex-Schneiderian papilloma, the second is morphologically most consistent with the, novel for this anatomic location, diagnosis of "invasive stratified mucin producing carcinoma" (ISMC), pointing to an analogy to similar, high-risk HPV-driven malignancies recently described in the gynecologic (GYN) and genitourinary (GU) areas. Both tumors, despite their mucoepidermoid-like features had no connection to salivary glands and lacked the MAML2 translocation typical of salivary gland MEC, pointing to a mucosal/non-salivary gland origin. Using these two carcinomas as examples, we attempt to address questions related to: (a) the histological distinction between MEC, adenosquamous carcinoma, and ISMC, (b) similarities and differences between these histological entities in mucosal sites versus morphologically similar salivary gland tumors, and (c) the role of HPV in these tumors.

Site-specific development and progressive maturation of human tissue-resident memory T cells over infancy and childhood

Infancy and childhood are critical life stages for generating immune memory to protect against pathogens; however, the timing, location, and pathways for memory development in humans remain elusive. Here, we investigated Tcells in mucosal sites, lymphoid tissues, and blood from 96 pediatric donors aged 0-10 years using phenotypic, functional, and transcriptomic profiling. Our results revealed that memory Tcells preferentially localized in the intestines and lungs during infancy and accumulated more rapidly in mucosal sites compared with blood and lymphoid organs, consistent with site-specific antigen exposure. Early life mucosal memory Tcells exhibit distinct functional capacities and stem-like transcriptional profiles. In later childhood, they progressively adopt proinflammatory functions and tissue-resident signatures, coincident with increased Tcell receptor (TCR) clonal expansion in mucosal and lymphoid sites. Together, our findings identify staged development of memory Tcells targeted to tissues during the formative years, informing how we might promote and monitor immunity in children.

Acquisition and annotation in high resolution in vivo digital biopsy by confocal microscopy for diagnosis in oral precancer and cancer.

Scanned fibre endomicroscopes are full point-scanning confocal microscopes with submicron lateral resolution with an optical slice thickness thin enough to isolate individual cell layers, allow active positioning of the optical slice in the z-axis and collection of megapixel images. Here we present descriptive findings and a brief atlas of an acquisition and annotation protocol high resolution in vivo capture of oral mucosal pathology including oral squamous cell carcinoma and dysplasia using a fluorescence scanned fibre endomicroscope with 3 topical fluorescent imaging agents: fluorescein, acriflavine and PARPi-FL. Digital biopsy was successfully performed via an acquisition protocol in seventy-one patients presenting for investigation of oral mucosal abnormalities using a miniaturized, handheld scanned fibre endoscope. Multiple imaging agents were utilized and multiple time points sampled. Fifty-nine patients had a matched histopathology correlating in location with imaging. The images were annotated back to macrographic location using a purpose-built software, MouthMap™. Acquisition and annotation of cellular level resolved images was demonstrated with all 3 topical agents. Descriptive observations between clinically or histologically normal oral mucosa showed regular intranuclear distance, a regular nuclear profile and fluorescent homogeneity. This was dependent on the intraoral location and type of epithelium being observed. Key features of malignancy were a loss of intranuclear distance, disordered nuclear clustering and irregular nuclear fluorescence intensity and size. Perinuclear fluorescent granules were seen in the absence of irregular nuclear features in lichenoid inflammation. High resolution oral biopsy allows for painless and rapid capture of multiple mucosal sites, resulting in more data points to increase diagnostic precision. High resolution digital micrographs can be easily compared serially across multiple time points utilizing an annotation software. In the present study we have demonstrated realization of a high-resolution digital biopsy protocol of the oral mucosa for utility in the diagnosis of oral cancer and precancer..

PLGA nanoparticles containing Intimin-Flagellin fusion protein for E. coli O157:H7 nano-vaccine

Escherichia coli O157:H7 is a foodborne pathogen that can lead to severe gastrointestinal diseases in humans. Vaccination is a promising strategy for preventing E. coli O157:H7 infections, which offers socio-economic benefits and provides the possibility of stimulating both humoral and cellular immune responses at systemic and mucosal sites. In this study, we developed a needle-free vaccine candidate against E. coli O157:H7 using poly(lactic-co-glycolic acid) (PLGA) nanoparticles entrapping a chimeric Intimin-Flagellin (IF) protein. The IF protein was expressed and verified using SDS-PAGE and western blot analysis, with a yield of 1/7 mg/L and a molecular weight of approximately 70 kDa. The prepared nanoparticles showed uniformly shaped spherical particles in the 200-nm range, as confirmed by SEM and DLS analysis. Three different routes of vaccine administration were used, including intranasal, oral, and subcutaneous, and the groups vaccinated with NPs protein had a higher antibody response compared to those receiving free protein. Subcutaneous administration of IF-NPs resulted in the highest level of IgG antibody titer, while oral administration of IF-NPs produced the highest amount of IgA antibody titer. Finally, all mice in the nanoparticle- intranasal and oral administered groups challenged with 100LD50 survived, while all control mice died before day 5. Based on these findings, we conclude that the PLGA-encapsulated IF protein has the potential to serve as a promising needle-free vaccine candidate against E. coli O157:H7.

Synthetic Self-Adjuvanted Lipopeptide Vaccines Conferred Protection against Helicobacter pylori Infection.

Helicobacter pylori(H. pylori) colonizes the stomach epithelium of half the world's population and is responsible for various digestive diseases and even stomach cancer. Vaccine-mediated protection against H. pylori infection depends primarily on the specific mucosal and T-cell responses. In this study, the synthetic lipopeptide vaccines, Hp4 (Pam2 Cys modified UreB T-cell epitope) and Hp10 (Pam2 Cys modified CagA T/B cell combined epitope), not only induce the bone marrow derived dendritic cells (BMDCs) maturation by activating a variety of pattern-recognition receptors (PRRs) such as Toll-like receptor (TLR), Nod-like receptor (NLR), and retinoic acid-inducing gene (RIG) I-like receptor (RLR), and but also stimulate BMDCs to secret cytokines that have the potential to modulate T-cell activation and differentiation. Although intranasal immunization with Hp4 or Hp10 elicits robust epitope-specific T-cell responses in mice, only Hp10 confers protection against H. pylori infection, possibly due to the fact that Hp10 also induces substantial specific sIgA response at mucosal sites. Interestingly, Hp4 elevates the protective response against H. pylori infection of Hp10 when administrated in combination, characterized by better protective effect and enhanced specific T-cell and mucosal antibody responses. The results suggest that synthetic lipopeptide vaccines based on the epitopes derived from the protective antigens are promising candidates for protection against H. pylori infection.

Comparison of Closure versus Non-closure of Buccal Mucosal Graft Harvesting Site in Urethroplasty.

Background: Buccal mucosal graft is commonly used in substitution urethroplasty for the treatment of anterior urethral strictures. However, the optimal management of the donor site remains a topic of debate. This prospective study aimed to compare the outcomes and morbidity associated with closure versus non-closure of the buccal mucosal graft harvesting site in urethroplasty.
 Methodology: A total of 60 patients with anterior urethral strictures underwent buccal mucosal urethroplasty. The patients were divided into two groups: group A (non-closure, n=30) and group B (closure, n=30). Post-operative pain, perioral numbness, early return to diet, and swelling of the cheek were assessed and compared between the two groups. Pain was measured using the Visual Analog Scale (VAS), and statistical analysis was performed using appropriate tests.
 Results: The mean age of the patients was 35.73 ± 13.42 years, and the mean length of the stricture was 58.45 ± 26.87 mm. Post-operative pain scores measured through VAS were comparable between the closure and non-closure groups at different time points. Perioral numbness was observed in 43.33% of the non-closure group and 30.0% of the closure group (p=0.284). Early return to diet was reported in 40.0% of the non-closure group and 70.0% of the closure group (p=0.019). Swelling of the cheek was noted in 53.33% of the non-closure group and 33.33% of the closure group (p=0.118).
 Conclusion: This study suggests that non-closure of the buccal mucosal graft harvesting site in urethroplasty results in less post-operative pain, early return to diet, and lower perioral numbness. However, it is associated with increased swelling of the cheek. These findings support the use of non-closure as a viable alternative to closure of the buccal mucosal graft harvesting site. Further research with larger sample sizes and longer-term follow-up is warranted to validate these findings and assess additional outcomes such as stricture recurrence and cosmetic outcomes.

Intranasal administration of sodium nitroprusside augments antigen-specific mucosal and systemic antibody production in mice

The coronavirus disease 2019, i.e., the COVID-19 pandemic, caused by a highly virulent and transmissible pathogen, has profoundly impacted global society. One approach to combat infectious diseases caused by pathogenic microbes is using mucosal vaccines, which can induce antigen-specific immune responses at both the mucosal and systemic sites. Despite its potential, the clinical implementation of mucosal vaccination is hampered by the lack of safe and effective mucosal adjuvants. Therefore, developing safe and effective mucosal adjuvants is essential for the fight against infectious diseases and the widespread clinical use of mucosal vaccines. In this study, we demonstrated the potent mucosal adjuvant effects of intranasal administration of sodium nitroprusside (SNP), a known nitric oxide (NO) donor, in mice. The results showed that intranasal administration of ovalbumin (OVA) in combination with SNP induced the production of OVA-specific immunoglobulin A in the mucosa and increased serum immunoglobulin G1 levels, indicating a T helper-2 (Th2)-type immune response. However, an analog of SNP, sodium ferrocyanide, which does not generate NO, failed to show any adjuvant effects, suggesting the critical role of NO generation in activating an immune response. In addition, SNPs facilitated the delivery of antigens to the lamina propria, where antigen-presenting cells are located, when co-administered with antigens, and also transiently elicited the expression of interleukin-6, interleukin-1β, granulocyte colony-stimulating factor, C-X-C motif chemokine ligand 1, and C-X-C motif chemokine ligand 2 in nasal tissue. These result suggest that SNP is a dual-functional formulation with antigen delivery capabilities to the lamina propria and the capacity to activate innate immunity. In summary, these results demonstrate the ability of SNP to induce immune responses via an antigen-specific Th2-type response, making it a promising candidate for further development as a mucosal vaccine formulation against infectious diseases.

Clotrimazole-Loaded Borneol-Based In Situ Forming Gel as Oral Sprays for Oropharyngeal Candidiasis Therapy.

Oral candidiasis encompasses fungal infections of the tongue and other oral mucosal sites with fungal overgrowth and its invasion of superficial oral tissues. Borneol was assessed in this research as the matrix-forming agent of clotrimazole-loaded in situ forming gel (ISG) comprising clove oil as the co-active agent and N-methyl pyrrolidone (NMP) as a solvent. Their physicochemical properties, including pH, density, viscosity, surface tension, contact angle, water tolerance, gel formation, and drug release/permeation, were determined. Their antimicrobial activities were tested using agar cup diffusion. The pH values of clotrimazole-loaded borneol-based ISGs were in the range of 5.59-6.61, which are close to the pH of 6.8 of saliva. Increasing the borneol content in the formulation slightly decreased the density, surface tension, water tolerance, and spray angle but increased the viscosity and gel formation. The borneol matrix formation from NMP removal promoted a significantly (p < 0.05) higher contact angle of the borneol-loaded ISGs on agarose gel and porcine buccal mucosa than those of all borneol-free solutions. Clotrimazole-loaded ISG containing 40% borneol demonstrated appropriate physicochemical properties and rapid gel formation at microscopic and macroscopic levels. In addition, it prolonged drug release with a maximum flux of 370 µg·cm-2 at 2 days. The borneol matrix generated from this ISG obsentively controlled the drug penetration through the porcine buccal membrane. Most clotrimazole amounts still remained in formulation at the donor part and then the buccal membrane and receiving medium, repectively. Therefore, the borneol matrix extended the drug release and penetration through the buccal membrane efficiently. Some accumulated clotrimazole in tissue should exhibit its potential antifugal activity against microbes invading the host tissue. The other predominant drug release into the saliva of the oral cavity should influence the pathogen of oropharyngeal candidiasis. Clotrimazole-loaded ISG demonstrated efficacious inhibition of growth against S. aureus, E. coli, C. albicans, C. krusei, C. Lusitaniae, and C. tropicalis. Consequently, the clotrimazole-loaded ISG exhibited great potential as a drug delivery system for oropharyngeal candidiasis treatment by localized spraying.

A topical rectal douche product containing Q-Griffithsin does not disrupt the epithelial border or alter CD4+ cell distribution in the human rectal mucosa

To reduce HIV transmission, locally applied pre-exposure prophylaxis (PrEP) products for anorectal use will be important complements to oral and injectable PrEP products already available. It is critical to preserve an intact rectal epithelium and avoid an influx of mucosal HIV target cells with such product use. In this phase 1 clinical trial, we evaluated application of a topical rectal douche product containing Q-Griffithsin (Q-GRFT). Colorectal tissue samples were obtained via sigmoidoscopy at baseline, 1 and 24 h after single-dose exposure in 15 healthy volunteers. In situ staining for epithelial junction markers and CD4+ cells were assessed as an exploratory endpoint. A high-throughput, digitalized in situ imaging analysis workflow was developed to visualize and quantify these HIV susceptibility markers. We observed no significant differences in epithelial distribution of E-cadherin, desmocollin-2, occludin, claudin-1, or zonula occludens-1 when comparing the three timepoints or Q-GRFT versus placebo. There were also no differences in %CD4+ cells within the epithelium or lamina propria in any of these comparisons. In conclusion, the rectal epithelium and CD4+ cell distribution remained unchanged following topical application of Q-GRFT. In situ visualization of HIV susceptibility markers at mucosal sites could be useful to complement standard product safety assessments.

Mucosal anomalies in autoimmune bullous diseases

Mucosal anomalies are frequently seen in autoimmune bullous diseases, particularly in pemphigus vulgaris and mucous membrane pemphigoid. The blistering, erosions, ulceration or erythema may present anywhere on the oral mucosa, but also on other mucosal sites. A differential diagnosis is needed of (erosive) oral lichen planus, systemic autoimmune disease, inflammatory bowel diseases, chronic graft-versus-host disease, infectious causes, Behçet's syndrome and recurrent aphthous stomatitis. A quick diagnosis and initiation of adequate treatment are important because of the potential severity of the disease and to prevent complications due to cicatrization. Besides a biopsy for histopathological analysis, a perilesional biopsy for direct immunofluorescence microscopy and immunoserological tests are needed for diagnosis of pemphigus or pemphigoid. In addition to a mucosal biopsy, a biopsy for direct immunofluorescence of the skin can contribute to a diagnosis of a bullous disease. Besides topical corticosteroids, immunosuppressive treatment is often required for treating autoimmune bullous diseases, such as treatment with rituximab in patients with pemphigus.

Mucosal immunization with Lactiplantibacillus plantarum-displaying recombinant SARS-CoV-2 epitopes on the surface induces humoral and mucosal immune responses in mice

BackgroundThe use of probiotic lactic acid bacteria as a mucosal vaccine vector is considered a promising alternative compared to the use of other microorganisms because of its “Generally Regarded as Safe” status, its potential adjuvant properties, and its tolerogenicity to the host. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19), is highly transmissible and pathogenic. This study aimed to determine the potential of Lactiplantibacillus plantarum expressing SARS-CoV-2 epitopes as a mucosal vaccine against SARS-CoV-2.ResultsIn this study, the possible antigenic determinants of the spike (S1–1, S1–2, S1–3, and S1–4), membrane (ME1 and ME2), and envelope (E) proteins of SARS-CoV-2 were predicted, and recombinant L. plantarum strains surface-displaying these epitopes were constructed. Subsequently, the immune responses induced by these recombinant strains were compared in vitro and in vivo. Most surface-displayed epitopes induced pro-inflammatory cytokines [tumor necrosis factor alpha (TNF-α and interleukin (IL)-6] and anti-inflammatory cytokines (IL-10) in lipopolysaccharide-induced RAW 264.7, with the highest anti-inflammatory to pro-inflammatory cytokine ratio in the S1–1 and S1–2 groups, followed by that in the S1–3 group. When orally administered of recombinant L. plantarum expressing SARS-CoV-2 epitopes in mice, all epitopes most increased the expression of IL-4, along with induced levels of TNF-α, interferon-gamma, and IL-10, specifically in spike protein groups. Thus, the surface expression of epitopes from the spike S1 protein in L. plantarum showed potential immunoregulatory effects, suggesting its ability to potentially circumvent hyperinflammatory states relevant to monocyte/macrophage cell activation. At 35 days post immunization (dpi), serum IgG levels showed a marked increase in the S1–1, S1–2, and S1–3 groups. Fecal IgA levels increased significantly from 21 dpi in all the antigen groups, but the boosting effect after 35 dpi was explicitly observed in the S1–1, S1–2, and S1–3 groups. Thus, the oral administration of SARS-CoV-2 antigens into mice induced significant humoral and mucosal immune responses.ConclusionThis study suggests that L. plantarum is a potential vector that can effectively deliver SARS-CoV-2 epitopes to intestinal mucosal sites and could serve as a novel approach for SARS-CoV-2 mucosal vaccine development.

The Interplay between Candida albicans, Vaginal Mucosa, Host Immunity and Resident Microbiota in Health and Disease: An Overview and Future Perspectives

Vulvovaginal candidiasis (VVC), which is primarily caused by Candida albicans, is an infection that affects up to 75% of all reproductive-age women worldwide. Recurrent VVC (RVVC) is defined as &gt;3 episodes per year and affects nearly 8% of women globally. At mucosal sites of the vagina, a delicate and complex balance exists between Candida spp., host immunity and local microbial communities. In fact, both immune response and microbiota composition play a central role in counteracting overgrowth of the fungus and maintaining homeostasis in the host. If this balance is perturbed, the conditions may favor C. albicans overgrowth and the yeast-to-hyphal transition, predisposing the host to VVC. To date, the factors that affect the equilibrium between Candida spp. and the host and drive the transition from C. albicans commensalism to pathogenicity are not yet fully understood. Understanding the host- and fungus-related factors that drive VVC pathogenesis is of paramount importance for the development of adequate therapeutic interventions to combat this common genital infection. This review focuses on the latest advances in the pathogenic mechanisms implicated in the onset of VVC and also discusses novel potential strategies, with a special focus on the use of probiotics and vaginal microbiota transplantation in the treatment and/or prevention of recurrent VVC.

AIM2 Promotes Epithelial Tuft Cell Development During Intestinal Type 2 Immune Responses

Abstract The epithelium performs a growing list of immunologically relevant functions at mucosal sites. Specialized epithelial cells of the intestine interact with the microbiota and lamina propria immune cells to regulate host defense, yet we know little about the molecular regulators linking these interactions during different immunological insults. Here, we found that the DNA sensor Absent in Melanoma 2 (AIM2) promotes development of epithelial tuft cells, which initiate intestinal type 2 immunity. Aim2-deficient intestinal epithelial organoids displayed reduced tuft cell gene signatures and numbers during treatment with the tuft cell-promoting cytokines IL-4 and IL-13. Tuft cells respond to the microbial metabolite succinate, leading to IL-13 production by type 2 innate lymphoid cells (ILC2s) and tuft cell hyperplasia in vivo. Succinate-fed Aim2 −/−mice displayed reduced tuft cells and IL13 expression compared to similarly treated wild type mice. Inflammasome-deficient Asc −/−mice and wild type mice displayed similar succinate-induced tuft cell profiles, suggesting AIM2 promotes tuft cell-ILC2 activation independently of its inflammasome function. RNA sequencing revealed Aim2 −/−intestinal organoids displayed diminished expression of O-linked N-acetylglucosamine transferase (OGT), which is required for IL13/STAT6-induced tuft cell expansion. In line with these findings, Aim2 −/−intestinal organoids and succinate-treated mice presented reduced OGT protein expression and STAT6 phosphorylation. These combined results reveal an novel role for AIM2 during type 2 intestinal inflammation in the intestine via promotion of tuft cell expansion, potentially through maintaining OGT-STAT6 signaling. Supported by grants from NIH (K22 CA212030) and the AZ Dept. of Health Services (AZBRC New Investigator Award).

Negative regulation of intestinal IgA by inflammatory cues in absence of infection.

Abstract Pathogen-specific IgA elicited at mucosal site is associated with a better immune protection upon vaccination. Although some effective mucosal vaccines are available and approved, the majority of current vaccines are administered intramuscularly or subcutaneously and were developed irrespectively of a particular site of infection. Vaccine adjuvants are selected in their capacity to elicit a strong inflammatory response. However, few studies show the effectiveness of FDA-approved adjuvants to mucosal vaccines. Here, we unveil the role of inflammatory pathways in their negative regulation of IgA production in murine intestines. We are currently studying and identifying the molecular and cellular pathways involved in the production of protective IgA antibodies in the intestines in absence of particular inflammatory cues. Indeed, in absence of some inflammatory signals, we observed an increase of free IgAs and IgA-bound bacteria in the intestines of mice in steady state. IgA levels were normal in the serum and the concentration of IgG antibodies were also normal in both the intestines and serum suggesting a skewed toward IgAs restricted to the intestines. Furthermore, we could show that this IgA production is for the most part T-dependent and involves the formation of germinal centers localized in the Peyer’s Patches. We are now developing vaccine models to elicit potent IgA-mediated protection in the aim to use these finding at our advantage to promote pathogen-specific intestinal responses through new designs of mucosal vaccines. We believe that developing mucosal vaccines that elicit a protective immunity through natural routes of infection hold the promise to act better, faster and safer against worldwide pathogenic threat. RWJF Grant Project # 826399 Moderna Research Fellowship

Imaging of lung resident memory CD8 +T cells reveals antigen-specific early protective responses during secondary infection

Abstract Tissue-resident memory T (T RM) cells are poised at mucosal sites to confer robust protection against secondary infection. In contrast to skin and intestine, lung T RMcells are short lived with a decline in number coinciding with a decline in protective immunity against subsequent heterosubtypic influenza infections. Despite this causal relationship, the localization, dynamics and function of lung Trm during secondary responses remains poorly defined. To address this, we used two-photon intravital microscopy of lungs to assess in real time the spatiotemporal dynamics of lung T RMcells during recall infection. At a memory time point, lung T RMcells (representing both CD103 +and CD103 −subsets) exhibit significantly lower motility and less displacement compared to an CD8 +effector T cells at an earlier time point post infection. Depletion of circulating CD8 +T cells using a low dose antibody administered systemically led to enrichment of lung T RMcells that exhibited slow to moderate speed scanning patterns. A fate mapping model for CD103 +T RMcells specifically revealed limited speed (&amp;lt;5 um/min) and patrolling behavior compared to CD103 −T RMcells. Within 24 hr of rechallenge, CD103 +T RMcells co-localized with infected cells and by 48 hr, lung-resident CD8 +T cells exhibit significantly lower motility with recruitment and clustering in infected regions in an antigen-specific manner. Protective immune responses by lung T RMcells is further accompanied by CXCR3-dependent migration of dendritic cells to draining lymph nodes as well as recruitment of inflammatory monocytes. Overall, these data unravel the spatiotemporal dynamics of lung T RMcells and interactions with recruited immune cells for early protective immunity. Supported by grants from NIH (GM134880, AI42767, AI114543, AI151183, AI167249, the Holden Cancer Center at the University of Iowa and its National Cancer Institute Award P30CA086862

ILC2-dependent induction of small intestinal memory and systemic mucosal resilience.

Abstract The IL-25-dependent ILC2-tuft cell circuit drives altered intestinal physiology in response to chronic helminth and protist infections. We optimized an induction regimen with recombinant IL-25 that resulted in long-lasting alterations of the small intestine, which we termed small intestinal memory. This state was characterized by expanded ILC2s and epithelial tuft cells, small intestinal lengthening, and enhanced resistance to helminth infection. ILC2s were necessary for the establishment of small intestinal memory, as this state was induced in Rag-deficient, but not Ragx gc-deficient mice or mice with deletion of IL-17RB on ILCs. ILC2s also maintained small intestinal memory, as this state was rapidly lost upon ILC2 ablation. Transcriptomic analysis revealed dynamic changes in ILC2 identities from IL-25-treated mice, demonstrative of innate memory. Strikingly, although these alterations were induced by IL-25, the ILC2 memory state was sustained in the absence of IL-25, tuft cells, or multiple alarmins like IL-33, TSLP, or IL-18. Finally, we found that small intestinal memory conferred resistance to bacterial and viral infections at distant mucosal sites and notably impacted the metabolic fitness of mice. The induction of enhanced mucosal resilience accompanied by increased metabolic fitness by IL-25 mimics the positive effects of chronic helminth infestation, revealing a mechanistic pathway that might underpin the widespread prevalence of these infections among vertebrates and that could be therapeutically adapted to improve mucosal health.