Search

Double negative T (DNT) cells are a unique subset of CD3 + TCRαβ + T lymphocytes that lack CD4, CD8, or NK1.1 expression and constitute 3-5% of the total T cell population in C57BL/6 mice. They have increasingly gained recognition for their novel roles in the immune system, especially under autoimmune conditions. Conventional machine learning approaches such as principal component analysis have been employed in single-cell RNA sequencing (scRNA-seq) analysis to characterize DNT cells. However, advanced deep learning models such as Single Cell Variational Inference (scVI) have the capability to capture nonlinear gene expression patterns in the sequencing data. In this study, employing the deep learning methodology, we have revealed novel markers for splenic DNT cells in C57BL/6 mice which were validated with flow cytometry analysis. We classified DNT cells into two subgroups, naïve DNT (nDNT) cells differentiated by the expression of Ly6C and activated DNT (aDNT) cells differentiated by the expression of MHC-II. A prior study had predicted elevated expression of CD137/4-1BB encoded by Tnfrsf9 in nDNT cells; however, our analysis predicted and validated that CD137 was a marker for aDNT cells instead of nDNT cells. Innovatively, our data also identified CD30 encoded by Tnfrsf8 and CD153/CD30L encoded by Tnfsf8 as additional markers for aDNT cells. In addition, we classified three subgroups in nDNT cells and two subgroups in aDNT cells. Our scVI analysis suggested, and flow cytometry analysis confirmed, that Ly49G2 encoded by Slamf7 was a marker for the nDNT0 subgroup. Importantly, we validated that MHC-II was indeed expressed by a subset of human DNT cells suggesting the presence of a human aDNT population. Furthermore, we found increased expression of CD30, CD153, and CD137 on aDNT cells in MRL/lpr mice compared to those in C57BL/6 mice suggesting potential pathogenic roles of these molecules in autoimmunity. Together, our comprehensive analysis has uncovered and validated novel markers for different subpopulations of DNT cells that can be used in the phenotypic and/or functional characterization of these relatively rare cells in health and disease., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Introduction: Leaky gut has been linked to autoimmune disorders including lupus. We previously reported upregulation of anti-flagellin antibodies in the blood of lupus patients and lupus-prone mice, which led to our hypothesis that a leaky gut drives lupus through bacterial flagellin-mediated activation of toll-like receptor 5 (TLR5)., Methods: We created MRL/ lpr mice with global Tlr5 deletion through CRISPR/Cas9 and investigated lupus-like disease in these mice., Result: Contrary to our hypothesis that the deletion of Tlr5 would attenuate lupus, our results showed exacerbation of lupus with Tlr5 deficiency in female MRL/ lpr mice. Remarkably higher levels of proteinuria were observed in Tlr5 -/- MRL/ lpr mice suggesting aggravated glomerulonephritis. Histopathological analysis confirmed this result, and Tlr5 deletion significantly increased the deposition of IgG and complement C3 in the glomeruli. In addition, Tlr5  deficiency significantly increased renal infiltration of Th17 and activated cDC1 cells. Splenomegaly and lymphadenopathy were also aggravated in Tlr5 -/- MRL/ lpr mice suggesting impact on lymphoproliferation. In the spleen, significant decreased frequencies of regulatory lymphocytes and increased germinal centers were observed with Tlr5 deletion. Notably, Tlr5 deficiency did not change host metabolism or the existing leaky gut; however, it significantly reshaped the fecal microbiota., Conclusion: Global deletion of Tlr5 exacerbates lupus-like disease in MRL/ lpr mice. Future studies will elucidate the underlying mechanisms by which Tlr5 deficiency modulates host-microbiota interactions to exacerbate lupus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Alajoleen, Oakland, Estaleen, Shakeri, Lu, Appiah, Sun, Neumann, Kawauchi, Cecere, McMillan, Reilly and Luo.)

Metabolic products of the microbiota can alter hematopoiesis. However, the contribution and site of action of bile acids is poorly understood. Here, we demonstrate that the secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA), increase bone marrow myelopoiesis. Treatment of bone marrow cells with DCA and LCA preferentially expanded immunophenotypic and functional colony-forming unit-granulocyte and macrophage (CFU-GM) granulocyte-monocyte progenitors (GMPs). DCA treatment of sorted hematopoietic stem and progenitor cells (HSPCs) increased CFU-GMs, indicating that direct exposure of HSPCs to DCA sufficed to increase GMPs. The vitamin D receptor (VDR) was required for the DCA-induced increase in CFU-GMs and GMPs. Single-cell RNA sequencing revealed that DCA significantly upregulated genes associated with myeloid differentiation and proliferation in GMPs. The action of DCA on HSPCs to expand GMPs in a VDR-dependent manner suggests microbiome-host interactions could directly affect bone marrow hematopoiesis and potentially the severity of infectious and inflammatory disease., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Clostridioides difficile infection (CDI) is the most common hospital-acquired infection in the United States. Antibiotic-induced dysbiosis is the primary cause of susceptibility, and fecal microbiota transplantation (FMT) has emerged as an effective therapy for recurrence. We previously demonstrated in the mouse model of CDI that antibiotic-induced dysbiosis reduced colonic expression of interleukin 25 (IL-25) and that FMT protected in part by restoring IL-25 signaling. Here, we conducted a prospective study in humans to test if FMT induced IL-25 expression in the colons of patients with recurrent CDI (rCDI). Colonic biopsy specimens and blood were collected at the time of FMT and 60 days later. Colon biopsy specimens were analyzed for IL-25 protein levels, total tissue transcriptome, and epithelium-associated microbiota before and after FMT, and peripheral immune cells were immunophenotyped. FMT increased alpha diversity of the colonic microbiota and levels of IL-25 in colonic tissue. In addition, FMT increased expression of homeostatic genes and repressed inflammatory genes. Finally, circulating Th17 cells were decreased post-FMT. The increase in levels of the cytokine IL-25 accompanied by decreased inflammation is consistent with FMT acting in part to protect from recurrent CDI via restoration of commensal activation of type 2 immunity. IMPORTANCE Fecal microbiota transplantation (FMT) is an effective treatment for C. difficile infection for most patients; however, introducing a complex mixture of microbes also has had unintended consequences for some patients. Attempts to create a standardized probiotic therapeutic that recapitulates the efficacy of FMT have been unsuccessful to date. We sought to understand what immune markers are changed in patients undergoing FMT to treat recurrent C. difficile infection and identified an immune signaling molecule, IL-25, that was restored by FMT. This finding indicates that adjunctive therapy with IL-25 could be useful in treating C. difficile infection.

Amebiasis is a neglected tropical disease caused by Entamoeba histolytic a. Although the disease burden varies geographically, amebiasis is estimated to account for some 55,000 deaths and millions of infections globally per year. Children and travelers are among the groups with the greatest risk of infection. There are currently no licensed vaccines for prevention of amebiasis, although key immune correlates for protection have been proposed from observational studies in humans. We previously described the development of a liposomal adjuvant formulation containing two synthetic TLR ligands (GLA and 3M-052) that enhanced antigen-specific fecal IgA, serum IgG2a, a mixed IFNγ and IL-17A cytokine profile from splenocytes, and protective efficacy following intranasal administration with the LecA antigen. By applying a statistical design of experiments (DOE) and desirability function approach, we now describe the optimization of the dose of each vaccine formulation component (LecA, GLA, 3M-052, and liposome) as well as the excipient composition (acyl chain length and saturation; PEGylated lipid:phospholipid ratio; and presence of antioxidant, tonicity, or viscosity agents) to maximize desired immunogenicity characteristics while maintaining physicochemical stability. This DOE/desirability index approach led to the identification of a lead candidate composition that demonstrated immune response durability and protective efficacy in the mouse model, as well as an assessment of the impact of each active vaccine formulation component on protection. Thus, we demonstrate that both GLA and 3M-052 are required for statistically significant protective efficacy. We also show that immunogenicity and efficacy results differ in female vs male mice, and the differences appear to be at least partly associated with adjuvant formulation composition., Competing Interests: MT is an employee of 3M and 3M-052 is an asset of 3M’s. WP is a consultant for TechLab, Inc. and in addition receives royalties for amebiasis diagnostics that are donated in their entirety to the American Society of Tropical Medicine and Hygiene. KP is an employee of TechLab, Inc. and amebiasis diagnostics are an asset of TechLab’s. CF, RK, SS, HL, IL, EL, JG, SL, AK, and SR are employees of IDRI, which owns assets including patents and patent applications involving formulations of GLA and 3M-052 including what is represented in this article. MA, WP, CF, and SL are inventors on patent/patent application(s) involving the vaccine formulations represented in this article. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Abhyankar, Orr, Kinsey, Sivananthan, Nafziger, Oakland, Young, Farr, Uddin, Leslie, Burgess, Liang, De Lima, Larson, Guderian, Lin, Kahn, Ghosh, Reed, Tomai, Pedersen, Petri and Fox.)

The microbiome provides resistance to infection. However, the underlying mechanisms are poorly understood. We demonstrate that colonization with the intestinal bacterium Clostridium scindens protects from Entamoeba histolytica colitis via innate immunity. Introduction of C. scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E. histolytica. Introduction of C. scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone marrow from C. scindens-colonized mice into naive mice protected against amebic colitis and increased intestinal neutrophils. Children without E. histolytica diarrhea also had a higher abundance of Lachnoclostridia. Lachnoclostridia C. scindens can metabolize the bile salt cholate, so we measured deoxycholate and discovered that it was increased in the sera of C. scindens-colonized specific pathogen-free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone increased GMPs and provided protection from amebiasis. We elucidated a mechanism by which C. scindens and the microbially metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with E. histolytica.

Giardia lamblia is a common intestinal parasitic infection that although often acutely asymptomatic, is associated with debilitating chronic intestinal and extra-intestinal sequelae. In previously healthy adults, a primary sporadic Giardia infection can lead to gut dysfunction and fatigue. These symptoms correlate with markers of inflammation that persist well after the infection is cleared. In contrast, in endemic settings, first exposure occurs in children who are frequently malnourished and also co-infected with other enteropathogens. In these children, Giardia rarely causes symptoms and associates with several decreased markers of inflammation. Mechanisms underlying these disparate and potentially enduring outcomes following Giardia infection are not presently well understood. A body of work suggests that the outcome of experimental Giardia infection is influenced by the nutritional status of the host. Here, we explore the consequences of experimental Giardia infection under conditions of protein sufficiency or deficiency on cytokine responses of ex vivo bone marrow derived dendritic cells (BMDCs) to endotoxin stimulation. We show that BMDCs from Giardia- challenged mice on a protein sufficient diet produce more IL-23 when compared to uninfected controls whereas BMDCs from Giardia challenged mice fed a protein deficient diet do not. Further, in vivo co-infection with Giardia attenuates robust IL-23 responses in endotoxin-stimulated BMDCs from protein deficient mice harboring enteroaggregative Escherichia coli. These results suggest that intestinal Giardia infection may have extra-intestinal effects on BMDC inflammatory cytokine production in a diet dependent manner, and that Giardia may influence the severity of the innate immune response to other enteropathogens. This work supports recent findings that intestinal microbial exposure may have lasting influences on systemic inflammatory responses, and may provide better understanding of potential mechanisms of post-infectious sequelae and clinical variation during Giardia and enteropathogen co-infection., Competing Interests: The authors have declared that no competing interests exist.

Mechanosensation is fundamentally important for the abilities of an organism to experience touch, hear sounds, and maintain balance. Caenorhabditis elegans is a powerful system for studying mechanosensation as this worm is well suited for in vivo functional imaging of neurons. Many years of research using labor-intensive methods have generated a wealth of knowledge about mechanosensation in C. elegans, and the recent microfluidic-based platforms continue to push the boundary for this field. However, developmental aspects of sensory biology, including mechanosensation, are still not fully understood. One current bottleneck is the difficulty in assaying larvae because they are much smaller than adult worms. Microfluidic devices with features small enough for larvae, especially actuators for the delivery of mechanical stimulation, are difficult to design and fabricate. Here, we present a series of automatic microfluidic platforms that allow for in vivo functional imaging of C. elegans responding to controlled mechanical stimulation at different developmental stages. Using a novel fabrication method, we designed highly deformable pneumatically actuated on-chip structures that can deliver mechanical stimulation to larval worms. The PDMS actuator allows for quantitatively controlled mechanical stimulation of both gentle and harsh touch neurons, by simply changing the actuation pressure, which makes this device easily translatable to other labs. We validated the design and utility of our systems with studies of the functional role of mechanosensory neurons in developing worms; we showed that gentle and harsh touch neurons function similarly in early larvae as they do in the adult stage, which would not have been possible previously. Finally, we investigated the effect of a sleep-like state on neuronal responses by imaging C. elegans in the lethargus state.