Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome.

Hundreds of microbiota genes are associated with host biology/disease. Unraveling the causal contribution of a microbiota gene to host biology remains difficult because many are encoded by nonmodel gut commensals and not genetically targetable. A general approach to identify their gene transfer methodology and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. We developed a pipeline that identifies the gene transfer methods for multiple nonmodel microbes spanning five phyla, and we demonstrated the utility of their genetic tools by modulating microbiome-derived short-chain fatty acids and bile acids in vitro and in the host. In a proof-of-principle study, by deleting a commensal gene for bile acid synthesis in a complex microbiome, we discovered an intriguing role of this gene in regulating colon inflammation. This technology will enable genetically engineering the nonmodel gut microbiome and facilitate mechanistic dissection of microbiota-host interactions.

Gut-innervating nociceptors regulate the intestinal microbiota to promote tissue protection.

Nociceptive pain is a hallmark of many chronic inflammatory conditions including inflammatory bowel diseases (IBDs); however, whether pain-sensing neurons influence intestinal inflammation remains poorly defined. Employing chemogenetic silencing, adenoviral-mediated colon-specific silencing, and pharmacological ablation of TRPV1+ nociceptors, we observed more severe inflammation and defective tissue-protective reparative processes in a murine model of intestinal damage and inflammation. Disrupted nociception led to significant alterations in the intestinal microbiota and a transmissible dysbiosis, while mono-colonization of germ-free mice with Gram+Clostridium spp. promoted intestinal tissue protection through a nociceptor-dependent pathway. Mechanistically, disruption of nociception resulted in decreased levels of substance P, and therapeutic delivery of substance P promoted tissue-protective effects exerted by TRPV1+ nociceptors in a microbiota-dependent manner. Finally, dysregulated nociceptor gene expression was observed in intestinal biopsies from IBD patients. Collectively, these findings indicate an evolutionarily conserved functional link between nociception, the intestinal microbiota, and the restoration of intestinal homeostasis.

Nutritional regulation of microbiota-derived metabolites: Implications for immunity and inflammation.

Nutrition profoundly shapes immunity and inflammation across the lifespan of mammals, from pre- and post-natal periods to later life. Emerging insights into diet-microbiota interactions indicate that nutrition has a dominant influence on the composition-and metabolic output-of the intestinal microbiota, which in turn has major consequences for host immunity and inflammation. Here, we discuss recent findings that support the concept that dietary effects on microbiota-derived metabolites potently alter immune responses in health and disease. We discuss how specific dietary components and metabolites can be either pro-inflammatory or anti-inflammatory in a context- and tissue-dependent manner during infection, chronic inflammation, and cancer. Together, these studies emphasize the influence of diet-microbiota crosstalk on immune regulation that will have a significant impact on precision nutrition approaches and therapeutic interventions for managing inflammation, infection, and cancer immunotherapy.

Inulin fibre promotes microbiota-derived bile acids and type 2 inflammation.

Dietary fibres can exert beneficial anti-inflammatory effects through microbially fermented short-chain fatty acid metabolites^1,2, although the immunoregulatory roles of most fibre diets and their microbiota-derived metabolites remain poorly defined. Here, using microbial sequencing and untargeted metabolomics, we show that a diet of inulin fibre alters the composition of the mouse microbiota and the levels of microbiota-derived metabolites, notably bile acids. This metabolomic shift is associated with type 2 inflammation in the intestine and lungs, characterized by IL-33 production, activation of group 2 innate lymphoid cells and eosinophilia. Delivery of cholic acid mimics inulin-induced type 2 inflammation, whereas deletion of the bile acid receptor farnesoid X receptor diminishes the effects of inulin. The effects of inulin are microbiota dependent and were reproduced in mice colonized with human-derived microbiota. Furthermore, genetic deletion of a bile-acid-metabolizing enzyme in one bacterial species abolishes the ability of inulin to trigger type 2 inflammation. Finally, we demonstrate that inulin enhances allergen- and helminth-induced type 2 inflammation. Taken together, these data reveal that dietary inulin fibre triggers microbiota-derived cholic acid and type 2 inflammation at barrier surfaces with implications for understanding the pathophysiology of allergic inflammation, tissue protection and host defence.

Non-redundant functions of group 2 innate lymphoid cells.

Protective immunity relies on the interplay of innate and adaptive immune cells with complementary and redundant functions. Innate lymphoid cells (ILCs) have recently emerged as tissue-resident, innate mirror images of the T cell system, with which they share lineage-specifying transcription factors and effector machinery1. Located at barrier surfaces, ILCs are among the first responders against invading pathogens and thus could potentially determine the outcome of the immune response2. However, so far it has not been possible to dissect the unique contributions of ILCs to protective immunity owing to limitations in specific targeting of ILC subsets. Thus, all of the available data have been generated either in mice lacking the adaptive immune system or with tools that also affect other immune cell subsets. In addition, it has been proposed that ILCs might be dispensable for a proper immune response because other immune cells could compensate for their absence3-7. Here we report the generation of a mouse model based on the neuromedin U receptor 1 (Nmur1) promoter as a driver for simultaneous expression of Cre recombinase and green fluorescent protein, which enables gene targeting in group 2 ILCs (ILC2s) without affecting other innate and adaptive immune cells. Using Cre-mediated gene deletion of Id2 and Gata3 in Nmur1-expressing cells, we generated mice with a selective and specific deficiency in ILC2s. ILC2-deficient mice have decreased eosinophil counts at steady state and are unable to recruit eosinophils to the airways in models of allergic asthma. Further, ILC2-deficient mice do not mount an appropriate immune and epithelial type 2 response, resulting in a profound defect in worm expulsion and a non-protective type 3 immune response. In total, our data establish non-redundant functions for ILC2s in the presence of adaptive immune cells at steady state and during disease and argue for a multilayered organization of the immune system on the basis of a spatiotemporal division of labour.

Epithelial-neuronal-immune cell interactions: Implications for immunity, inflammation, and tissue homeostasis at mucosal sites.

The epithelial lining of the respiratory tract and intestine provides a critical physical barrier to protect host tissues against environmental insults, including dietary antigens, allergens, chemicals, and microorganisms. In addition, specialized epithelial cells communicate directly with hematopoietic and neuronal cells. These epithelial-immune and epithelial-neuronal interactions control host immune responses and have important implications for inflammatory conditions associated with defects in the epithelial barrier, including asthma, allergy, and inflammatory bowel diseases. In this review, we discuss emerging research that identifies the mechanisms and impact of epithelial-immune and epithelial-neuronal cross talk in regulating immunity, inflammation, and tissue homeostasis at mucosal barrier surfaces. Understanding the regulation and impact of these pathways could provide new therapeutic targets for inflammatory diseases at mucosal sites.

Contrast agents for x-ray luminescence computed tomography.

Imaging probes are an important consideration for any type of contrast agent-based imaging method. X-ray luminescence imaging (XLI) and x-ray luminescence computed tomography (XLCT) are both contrast agent-based imaging methods that employ x-ray excitable scintillating imaging probes that emit light to be measured for optical imaging. In this work, we compared the performance of several select imaging probes, both commercial and self-synthesized, for application in XLI/XLCT imaging. Commercially available cadmium telluride quantum dots (CdTe QDs) and europium-doped gadolinium oxysulfide (GOS:Eu) microphosphor as well as synthesized NaGdF4 nanophosphors doped with either europium or terbium were compared through their x-ray luminescence emission spectra, luminescence intensity, and also by performing XLCT scans using phantoms embedded with each of the imaging probes. Each imaging probe displayed a unique emission spectrum that was ideal for deep-tissue optical imaging. In terms of luminescence intensity, due to the large particle size, GOS:Eu had the brightest emission, followed by NaGdF4:Tb, NaGdF4:Eu, and finally the CdTe QDs. Lastly, XLCT scans showed that each imaging probe could be reconstructed with good shape and location accuracy.

Microbiota metabolism of intestinal amino acids impacts host nutrient homeostasis and physiology.

The intestine and liver are thought to metabolize dietary nutrients and regulate host nutrient homeostasis. Here, we find that the gut microbiota also reshapes the host amino acid (aa) landscape via efficiently metabolizing intestinal aa. To identify the responsible microbes/genes, we developed a metabolomics-based assay to screen 104 commensals and identified candidates that efficiently utilize aa. Using genetics, we identified multiple responsible metabolic genes in phylogenetically diverse microbes. By colonizing germ-free mice with the wild-type strain and their isogenic mutant deficient in individual aa-metabolizing genes, we found that these genes regulate the availability of gut and circulatory aa. Notably, microbiota genes for branched-chain amino acids (BCAAs) and tryptophan metabolism indirectly affect host glucose homeostasis via peripheral serotonin. Collectively, at single-gene level, this work characterizes a microbiota-encoded metabolic activity that affects host nutrient homeostasis and provides a roadmap to interrogate microbiota-dependent activity to improve human health.

Dietary fiber is a critical determinant of pathologic ILC2 responses and intestinal inflammation.

Innate lymphoid cells (ILCs) can promote host defense, chronic inflammation, or tissue protection and are regulated by cytokines and neuropeptides. However, their regulation by diet and microbiota-derived signals remains unclear. We show that an inulin fiber diet promotes Tph1-expressing inflammatory ILC2s (ILC2INFLAM) in the colon, which produce IL-5 but not tissue-protective amphiregulin (AREG), resulting in the accumulation of eosinophils. This exacerbates inflammation in a murine model of intestinal damage and inflammation in an ILC2- and eosinophil-dependent manner. Mechanistically, the inulin fiber diet elevated microbiota-derived bile acids, including cholic acid (CA) that induced expression of ILC2-activating IL-33. In IBD patients, bile acids, their receptor farnesoid X receptor (FXR), IL-33, and eosinophils were all upregulated compared with controls, implicating this diet-microbiota-ILC2 axis in human IBD pathogenesis. Together, these data reveal that dietary fiber-induced changes in microbial metabolites operate as a rheostat that governs protective versus pathologic ILC2 responses with relevance to precision nutrition for inflammatory diseases.

The ChAT-acetylcholine pathway promotes group 2 innate lymphoid cell responses and anti-helminth immunity.

Group 2 innate lymphoid cells (ILC2s) reside in multiple tissues, including lymphoid organs and barrier surfaces, and secrete type 2 cytokines including interleukin-5 (IL-5), IL-9, and IL-13. These cells participate in multiple physiological processes including allergic inflammation, tissue repair, metabolic homeostasis, and host defense against helminth infections. Recent studies indicate that neurotransmitters and neuropeptides can play an important role in regulating ILC2 responses; however, the mechanisms that underlie these processes in vivo remain incompletely defined. Here, we identify that activated ILC2s up-regulate choline acetyltransferase (ChAT)-the enzyme responsible for the biosynthesis of acetylcholine (ACh)-after infection with the helminth parasite Nippostrongylus brasiliensis or treatment with alarmins or cytokines including IL-25, IL-33, and thymic stromal lymphopoietin (TSLP). ILC2s also express acetylcholine receptors (AChRs), and ACh administration promotes ILC2 cytokine production and elicits expulsion of helminth infection. In accordance with this, ChAT deficiency in ILC2s leads to defective ILC2 responses and impaired immunity against helminth infection. Together, these results reveal a previously unrecognized role of the ChAT-ACh pathway in promoting type 2 innate immunity to helminth infection.

Focused x-ray luminescence imaging system for small animals based on a rotary gantry.

SIGNIFICANCE: The ability to detect and localize specific molecules through tissue is important for elucidating the molecular basis of disease and treatment. Unfortunately, most current molecular imaging tools in tissue either lack high spatial resolution (e.g., diffuse optical fluorescence tomography or positron emission tomography) or lack molecular sensitivity (e.g., micro-computed tomography, µCT). X-ray luminescence imaging emerged about 10 years ago to address this issue by combining the molecular sensitivity of optical probes with the high spatial resolution of x-ray imaging through tissue. In particular, x-ray luminescence computed tomography (XLCT) has been demonstrated as a powerful technique for the high-resolution imaging of deeply embedded contrast agents in three dimensions (3D) for small-animal imaging. AIM: To facilitate the translation of XLCT for small-animal imaging, we have designed and built a small-animal dedicated focused x-ray luminescence tomography (FXLT) scanner with a µCT scanner, synthesized bright and biocompatible nanophosphors as contrast agents, and have developed a deep-learning-based reconstruction algorithm. APPROACH: The proposed FXLT imaging system was designed using computer-aided design software and built according to specifications. NaGdF4 nanophosphors doped with europium or terbium were synthesized with a silica shell for increased biocompatibility and functionalized with biotin. A deep-learning-based XLCT image reconstruction was also developed based on the residual neural network as a data synthesis method of projection views from few-view data to enhance the reconstructed image quality. RESULTS: We have built the FXLT scanner for small-animal imaging based on a rotational gantry. With all major imaging components mounted, the motor controlling the gantry can be used to rotate the system with a high accuracy. The synthesized nanophosphors displayed distinct x-ray luminescence emission, which enables multi-color imaging, and has successfully been bound to streptavidin-coated substrates. Lastly, numerical simulations using the proposed deep-learning-based reconstruction algorithm has demonstrated a clear enhancement in the reconstructed image quality. CONCLUSIONS: The designed FXLT scanner, synthesized nanophosphors, and deep-learning-based reconstruction algorithm show great potential for the high-resolution molecular imaging of small animals.

Development of immunoglobulin M memory to both a T-cell-independent and a T-cell-dependent antigen following infection with Vibrio cholerae O1 in Bangladesh.

Vibrio cholerae O1 can cause severe watery diarrhea that can be life-threatening without treatment. Infection results in long-lasting protection against subsequent disease. Development of memory B cells of the immunoglobulin G (IgG) and IgA isotypes to V. cholerae O1 antigens, including serotype-specific lipopolysaccharide (LPS) and the B subunit of cholera toxin (CTB), after cholera infection has been demonstrated. Memory B cells of the IgM isotype may play a role in long-term protection, particularly against T-cell-independent antigens, but IgM memory has not been studied in V. cholerae O1 infection. Therefore, we assayed acute- and convalescent-phase blood samples from cholera patients for the presence of memory B cells that produce cholera antigen-specific IgM antibody upon polyclonal stimulation in in vitro culture. We also examined the development of serological and antibody-secreting cell responses following infection. Subjects developed significant IgM memory responses by day 30 after infection, both to the T-cell-independent antigen LPS and to the T-cell-dependent antigen CTB. No significant corresponding elevations in plasma IgM antibodies or circulating IgM antibody-secreting cells to CTB were detected. In 17 subjects followed to day 90 after infection, significant persistence of elevated IgM memory responses was not observed. The IgM memory response to CTB was negatively correlated with the IgG plasma antibody response to CTB, and there was a trend toward negative correlation between the IgM memory and IgA plasma antibody responses to LPS. We did not observe an association between the IgM memory response to LPS and the vibriocidal titer.

Transcutaneous Vaccination with Conjugate Typhoid Vaccine Vi-DT Induces Systemic, Mucosal, and Memory Anti-Polysaccharide Responses.

Transcutaneous vaccination can induce both mucosal and systemic immune responses. However, there are few data on anti-polysaccharide responses following transcutaneous vaccination of polysaccharides, despite the role that anti-polysaccharide responses play in protecting against intestinal mucosal and respiratory pathogens. Whether transcutaneous vaccination with a conjugate polysaccharide vaccine would be able to induce memory responses is also unknown. To address this, we transcutaneously vaccinated mice with virulence antigen (Vi) polysaccharide of Salmonella enterica serovar Typhi (the cause of typhoid fever), either in unconjugated or conjugated form (the latter as a Vi-DT conjugate). We also assessed the ability of the immunoadjuvant cholera toxin to impact responses following vaccination. We found that presenting Vi in a conjugate versus nonconjugate form transcutaneously resulted in comparable serum IgG responses but higher serum and lamina propria lymphocyte IgA anti-Vi responses, as well as increased IgG memory responses. The addition of immunoadjuvant did not further increase these responses; however, it boosted fecal IgA and serum IgG anti-Vi responses. Our results suggest that transcutaneous vaccination of a conjugate vaccine can induce systemic as well as enhanced mucosal and memory B-cell anti-polysaccharide responses.

Children with the Le(a+b-) blood group have increased susceptibility to diarrhea caused by enterotoxigenic Escherichia coli expressing colonization factor I group fimbriae.

Recent studies have shown that children with blood group A have increased susceptibility to enterotoxigenic Escherichia coli (ETEC) diarrhea and that Lewis blood group "a" antigen (Le(a)) may be a candidate receptor for ETEC colonization factor (CF) antigen I (CFA/I) fimbriae. Based on these findings, we have attempted to determine if children with the Le(a+b-) phenotype may be more susceptible to diarrhea caused by ETEC, in particular ETEC expressing CFA/I and related fimbriae of the CFA/I group, than Le(a-b+) children. To test this hypothesis, we have determined the Lewis antigen expression in 179 Bangladeshi children from a prospective birth cohort study in urban Dhaka in which ETEC expressing major CFs such as CFA/I, CS3, CS5, and CS6 was the most commonly isolated diarrhea pathogen during the first 2 years of life. The Lewis blood group phenotypes were determined by a dot blot immunoassay using saliva samples and by a tube agglutination test using fresh red blood cells. The results indicate that Le(a+b-) children more often had symptomatic than asymptomatic ETEC infections (P < 0.001), whereas symptomatic and asymptomatic ETEC infections were equally frequent in Le(a-b+) children. We also show that children with the Le(a+b-) blood type had significantly higher incidences of diarrhea caused by ETEC expressing fimbriae of the CFA/I group than Le(a-b+) children (P < 0.001). In contrast, we did not find any association between the Lewis blood group phenotype and diarrhea caused by ETEC expressing CS6 or rotavirus.

Memory T-cell responses to Vibrio cholerae O1 infection.

Vibrio cholerae O1 can cause diarrheal disease that may be life-threatening without treatment. Natural infection results in long-lasting protective immunity, but the role of T cells in this immune response has not been well characterized. In contrast, robust B-cell responses to V. cholerae infection have been observed. In particular, memory B-cell responses to T-cell-dependent antigens persist for at least 1 year, whereas responses to lipopolysaccharide, a T-cell-independent antigen, wane more rapidly after infection. We hypothesize that protective immunity is mediated by anamnestic responses of memory B cells in the gut-associated lymphoid tissue, and T-cell responses may be required to generate and maintain durable memory B-cell responses. In this study, we examined B- and T-cell responses in patients with severe V. cholerae infection. Using the flow cytometric assay of the specific cell-mediated immune response in activated whole blood, we measured antigen-specific T-cell responses using V. cholerae antigens, including the toxin-coregulated pilus (TcpA), a V. cholerae membrane preparation, and the V. cholerae cytolysin/hemolysin (VCC) protein. Our results show that memory T-cell responses develop by day 7 after infection, a time prior to and concurrent with the development of B-cell responses. This suggests that T-cell responses to V. cholerae antigens may be important for the generation and stability of memory B-cell responses. The T-cell proliferative response to VCC was of a higher magnitude than responses observed to other V. cholerae antigens.

MRGPR-mediated activation of local mast cells clears cutaneous bacterial infection and protects against reinfection.

Mast cells (MCs) are strategically distributed at barrier sites and prestore various immunocyte-recruiting cytokines, making them ideal targets for selective activation to treat peripheral infections. Here, we report that topical treatment with mastoparan, a peptide MC activator (MCA), enhances clearance of Staphylococcus aureus from infected mouse skins and accelerates healing of dermonecrotic lesions. Mastoparan functions by activating connective tissue MCs (CTMCs) via the MRGPRX2 (Mas-related G protein-coupled receptor member X2) receptor. Peripheral CTMC activation, in turn, enhances recruitment of bacteria-clearing neutrophils and wound-healing CD301b+ dendritic cells. Consistent with MCs playing a master coordinating role, MC activation also augmented migration of various antigen-presenting dendritic cells to draining lymph nodes, leading to stronger protection against a second infection challenge. MCAs therefore orchestrate both the innate and adaptive immune arms, which could potentially be applied to combat peripheral infections by a broad range of pathogens.

Necroptosis of infiltrated macrophages drives Yersinia pestis dispersal within buboes.

When draining lymph nodes become infected by Yersinia pestis (Y. pestis), a massive influx of phagocytic cells occurs, resulting in distended and necrotic structures known as buboes. The bubonic stage of the Y. pestis life cycle precedes septicemia, which is facilitated by trafficking of infected mononuclear phagocytes through these buboes. However, how Y. pestis convert these immunocytes recruited by host to contain the pathogen into vehicles for bacterial dispersal and the role of immune cell death in this context are unknown. We show that the lymphatic spread requires Yersinia outer protein J (YopJ), which triggers death of infected macrophages by downregulating a suppressor of receptor-interacting protein kinase 1-mediated (RIPK1-mediated) cell death programs. The YopJ-triggered cell death was identified as necroptotic, which released intracellular bacteria, allowing them to infect new neighboring cell targets. Dying macrophages also produced chemotactic sphingosine 1-phosphate, enhancing cell-to-cell contact, further promoting infection. This necroptosis-driven expansion of infected macrophages in buboes maximized the number of bacteria-bearing macrophages reaching secondary lymph nodes, leading to sepsis. In support, necrostatins confined bacteria within macrophages and protected mice from lethal infection. These findings define necrotization of buboes as a mechanism for bacterial spread and a potential target for therapeutic intervention.

Thalassemias in South Asia: clinical lessons learnt from Bangladesh.

Thalassemias are emerging as a global public health concern. Due to remarkable success in the reduction of childhood mortality by controlling infectious diseases in developing countries, thalassemias are likely to be a major public health concern in the coming decades in South Asia. Despite the fact that Bangladesh lies in the world's thalassemia belt, the information on different aspects (epidemiology, clinical course, mortality, complications and treatment outcomes) of thalassemias is lacking. In this comprehensive review, the aim is to to depict the epidemiological aspects of thalassemias, mutation profile and current treatment and management practices in the country by sharing the experience of dealing with 1178 cases over 2009-2014 time periods in a specialized thalassemia treatment centre. We have also discussed the preventative strategies of thalassemias from the context of Bangladesh which could be effective for other developing countries.

The ATPase domain of HscC (DnaK homolog) is essential for interfering sigma70 activity in E. coli.

HscC, a DnaK homolog in Escherichia coli, consists of adenosine triphosphatase (ATPase), substrate-binding and C-terminal domains. Overexpression of HscC markedly inhibits growth of host cell and reduces the sigma(70)-dependent promoter activity presumably by forming a complex with sigma(70). To identify the region(s) of HscC responsible for growth inhibition and complex formation with sigma(70), domain swapping experiments were carried out between DnaK and HscC. Thus the chimeric proteins carrying the ATPase domain of HscC and substrate-binding domains of either HscC or DnaK were found to inhibit the growth of the cell, reduce the sigma(70)-dependent promoter activity and form a complex with sigma(70). These results indicate that the ATPase domain of HscC rather than the substrate-binding domain is important for determining its functional specificity.

Study of avidity of antigen-specific antibody as a means of understanding development of long-term immunological memory after Vibrio cholerae O1 infection.

The avidity of antibodies to specific antigens and the relationship of avidity to memory B cell responses to these antigens have not been studied in patients with cholera or those receiving oral cholera vaccines. We measured the avidity of antibodies to cholera toxin B subunit (CTB) and Vibrio cholerae O1 lipopolysaccharide (LPS) in Bangladeshi adult cholera patients (n = 30), as well as vaccinees (n = 30) after administration of two doses of a killed oral cholera vaccine. We assessed antibody and memory B cell responses at the acute stage in patients or prior to vaccination in vaccinees and then in follow-up over a year. Both patients and vaccinees mounted CTB-specific IgG and IgA antibodies of high avidity. Patients showed longer persistence of these antibodies than vaccinees, with persistence lasting in patients up to day 270 to 360. The avidity of LPS-specific IgG and IgA antibodies in patients remained elevated up to 180 days of follow-up. Vaccinees mounted highly avid LPS-specific antibodies at day 17 (3 days after the second dose of vaccine), but the avidity waned rapidly to baseline by 30 days. We examined the correlation between antigen-specific memory B cell responses and avidity indices for both antigens. We found that numbers of CTB- and LPS-specific memory B cells significantly correlated with the avidity indices of the corresponding antibodies (P < 0.05; Spearman's ρ = 0.28 to 0.45). These findings suggest that antibody avidity after infection and immunization is a good correlate of the development and maintenance of memory B cell responses to Vibrio cholerae O1 antigens.