bluuebunny/crossref_metadata_2025 huggingface dataset:
Broad analysis of MTB-specific CD4 T cell responses reveals long lasting effect from previous active TB
Authors: Lindestam Arlehamn Cecilia S , Carpenter Chelsea , McKinney Denise M. , Makgotlho Edward , Rozot Virginie , Hanekom Willem , Peters Bjoern , Scriba Thomas J , Sette Alessandro
Published: January 2023

We have performed a side-by-side comparison of the immunogenicity of 11 antigens currently in candidate TB vaccines in clinical trials and known CD4 epitopes using PBMCs from healthy individuals with latent M. tuberculosis (MTB) infection (LTBI, n=42) or with prior active TB disease (PATB, n=21). The PATB samples were further divided based on time since TB diagnosis; less or more than 6 years ago. The epitopes identified in these cohorts were categorized into (1) maintained latency (LTBI/PATB ratio >2), (2) associated with continued control of MTB infection i.e. LTBI status, and (3) shared epitopes (LTBI/PATB ratio <2). Interestingly, the response to the maintained latency epitopes was significantly higher in LTBIs and PATBs with a longer time since diagnosis compared to PATB less than 6 years ago, but no difference was observed for the shared epitopes. This suggests that progression to active TB and/or chemotherapy is associated with loss of recognition of the maintained latency epitopes, with reactivity gradually regained several years after TB treatment, when these individuals may have become re-infected. Differential reactivity in these cohorts was also revealed at the antigen level. The biological relevance of this was further demonstrated by phenotypic differences of MTB-specific T cells in these cohorts. These results demonstrate that a large panel of epitopes is required to reveal differences in immune reactivity between LTBIs and PATB and suggest that previous active TB disease provokes a long lasting influence on the immune response.
Recruitment of myeloid cells into mycobacterial granulomas.
Authors: Herbath Melinda , Harding Jeffrey S. , Haskó György , Nagy András , Fábry Zsuzsanna , Sándor Mátyás
Published: January 2023

Granuloma formation is a hallmark of mycobacterial infection. The dominant cell types in granuloma are the blood monocyte derived macrophages and inflammatory dendritic cells that represent up to 80% of cells in these lesions. Using granuloma transplantation we demonstrated that in a week more than the third of these cells are replaced. Innate and cognate immunity against the bacteria induces a battery of chemokines that are important in this recruitment and MCP1-CCR2 is one of the main representatives of these pathways. We have also shown that cell death and death-induced extracellular ATP through P2X7R promote VEGF production in a subpopulation of granuloma macrophages and that is also important for myeloid cell recruitment through VEGFR1 displayed by blood monocytes. These factors affect granuloma size, numbers and the number of extravasated monocytes in the tissue around granulomas.

We further compare BCG-induced liver and Mtb-induced lung granulomas in wild type, hypoVEGF, macrophage VEGF production ablated, selective macrophage P2X7R deficient, and CCR2 deficient mice. Additionally to the size and frequency of granulomas we compare cell composition, the cytokines present and bacterial load in the lesions. These data clarify the role of immune response and cell death driven cell recruitment pathways in the granuloma maintenance.
NLRP3 inflammasome plays an important role in the innate immune response to Acinetobacter baumannii infection
Authors: Sukumaran Bindu , Dikshit Neha
Published: January 2023

Acinetobacter baumannii is an emerging multi- or pan-drug resistant nosocomial and community associated bacterium that is increasingly becoming a serious health threat world-wide. Pneumonia, bacteremia, urinary tract infections, and wound infections are some of the important clinical manifestations of A.baumannii infection. However, the innate immune pathways leading to various clinical outcomes associated with A. baumannii infections are not fully characterized yet. We identified that A. baumannii invades and survives within host cells and hypothesized that intracellular innate immune receptors might play a role in the host defense to A. baumannii infection. Inflammasomes are intracellular multiprotein complexes that play critical roles in innate immunity. But whether inflammasome pathway plays any role in the pathogenesis of A. baumannii infection is not studied yet. Through in vitro and in vivo murine pneumonia infection models, we demonstrate that the most extensively studied inflammasome, NLRP3, is an important player in the host defense and inflammatory response associated with A. baumannii infection. We show that A.baumannii infection primes as well as activates the NLRP3 inflammasome assembly in mouse bone marrow-derived macrophages. A.baumannii infection induced IL-1β/IL-18 production is entirely dependent upon ASC/NLRP3/Caspase-1 axis. A.baumannii activates NLRP3 inflammasome by various overlapping mechanisms such as ROS generation/Potassium ion imbalance. Thus we identified a previously unrecognized role for inflammasome components in the host innate immune response to A. baumannii infection and this information may provide alternative therapeutic approaches to tackle A. baumannii infections.
A TLR4-interacting surfactant protein-A-derived peptide improves host defense against Pseudomonas aeruginosa.
Authors: Awasthi Shanjana , Singh Bhupinder , Ramani Vijay , Awasthi Vibhudutta , Xie Jun , Kosanke Stanley
Published: January 2023

Interaction between surfactant protein-A (SP-A) and toll-like receptor (TLR4) plays a critical role in host defense. We have identified SPA4 peptide (amino acids: GDFRYSDGTPVNYTNWYRGE) from a TLR4-interacting region of SP-A. The SPA4 peptide binds to TLR4 and suppresses the secreted levels of inflammatory cytokines against Gram-negative bacterial lipopolysaccharide. In this work, we studied the host defense function of SPA4 peptide against Pseudomonas aeruginosa. We examined the binding of SPA4 peptide to cellular TLR4 and bacteria, and direct antibacterial activity against P. aeruginosa. Pro-phagocytic and anti-inflammatory activities of SPA4 peptide were investigated in nontransfected and TLR4-transfected dendritic cells and primary mouse alveolar macrophages. The biological activity of SPA4 peptide was then studied in a mouse model of P. aeruginosa lung infection. Our results demonstrate that the SPA4 peptide binds to TLR4, but does not interact with live bacteria. Correspondingly, the SPA4 peptide does not directly affect bacterial growth. The SPA4 peptide treatment induces the uptake and localization of bacteria in the phagolysosomes of immune cells. At the same time, the secreted levels of inflammatory cytokines are significantly suppressed. Over-expression of TLR4 augments the pro-phagocytic and anti-inflammatory activity of SPA4 peptide. Furthermore, treatment with SPA4 peptide reduces the bacterial burden, inflammatory cytokines, phosphorylation of p38 MAPK, p54 SAPK/JNK and NF-κB-p65, and alleviates lung edema and pathology in a mouse model of P. aeruginosa infection. Together, our results suggest that the immunomodulatory activity of SPA4 peptide can potentially help control infection.
Control of lupus nephritis by the gut microbiota
Authors: Luo Xin M , Mu Qinghui
Published: January 2023

Dysbiosis of gut microbiota promotes autoimmune disorders that damage organs outside the intestine. Here we report the role of gut microbiota in the pathogenesis of renal dysfunction in systemic lupus erythematosus. In a classical mouse model of lupus nephritis, MRL/lpr, we found marked depletion of Lactobacillus spp. in the gut microbiota. Increasing Lactobacilli in the gut improved renal function of these mice. Further studies revealed that MRL/lpr mice possessed a “leaky” gut, which was reversed by Lactobacilli. Lactobacillus treatment also decreased mucosal IL-6 and increased mucosal IL-10, thereby providing an anti-inflammatory environment in the gut. In addition, it increased IL-10 and decreased IgG2a in the circulation, the latter a major immune deposit in the kidney of MRL/lpr mice. Inside the kidney, Treg-Th17 balance was skewed towards Treg with Lactobacillus treatment. These effects of Lactobacilli were present in female and castrated male mice, but not in unaltered males, suggesting that beneficial bacteria in the gut attenuate lupus nephritis in a sex hormone-dependent manner.
Role of the innate immune system in systemic enterococcus dissemination and clearance
Authors: Chakraborty Rajrupa , Kommineni Sushma , Hayward Michael , Nelson Tamara , Kristich Christopher J , Salzman Nita H
Published: January 2023

Enterococci are gram-positive commensals of the mammalian intestinal tract and intrinsically resistant to broad-spectrum cephalosporin antibiotics. They proliferate in the gut and disseminate systemically causing infections in immunocompromised individuals and hospitalized patients undergoing cephalosporin therapy. Using a unique mouse model with stable intestinal colonization with a lab strain of Enterococcus faecalis (EF), we investigated the role of the immune system in EF dissemination and clearance from mice treated with cephalosporins. Ceftriaxone mediated depletion of indigenous microbiota was associated with expansion of EF throughout the gut, reduced levels of gut mucosal barrier components and eventually EF dissemination to peripheral organs. We observed that EF was not associated with the FACS sorted populations of lamina propria mononuclear phagocytes suggesting that these immune cells did not actively translocate EF across the intestinal mucosa. Mice cleared EF systemically by 2 weeks post-antibiotic treatment without evidence of adaptive immune response. Studies in Rag1−/− mice suggested that adaptive immune system was not essential in EF clearance or containment. Our observations support that intestinal containment and systemic clearance of EF are mediated by innate immune mechanisms. We predict that this reflects general mechanisms for host containment and management of the intestinal commensal microbiota.
Identification of ribosomal targets for protective CD4+ T cell responses against mycobacteria
Authors: Johnson Alison Joy , Kennedy Steven C , Lindestam Arlehamn Cecilia S , Hegde Subray S. , Blanchard John S. , Sette Alessandro , Chan John , Jacobs William R. , Porcelli Steven A.
Published: January 2023

Mycobacterium tuberculosis (Mtb) is a significant health burden, infecting one-third of the global population. A more efficacious vaccine is urgently needed for control and prevention of disease caused by Mtb. We previously reported a genetically modified strain of Mycobacterium smegmatis called IKEPLUS, which is a promising vaccine candidate due to its ability to generate an unprecedented level of bactericidal immunity against Mtb in mice. Protective immunity induced by IKEPLUS is dependent on antigen-specific CD4+ T cell memory, and we hypothesized that specificity of the CD4+ T cell response was a critical feature of this protection. Using in vitro assays of IFNγ production by splenocytes from immunized mice, we identified an immunogenic peptide within the mycobacterial RplJ protein encoded by the Rv0651 gene in mice immunized with IKEPLUS, but not in mice immunized with the currently available M. bovis BCG vaccine. This difference in the immune response generated by the two vaccine strategies led us to further investigate the immunogenicity of the mycobacterial ribosome for additional immunogenic targets. Screening a synthetic peptide library based on the sequences of the 57 mycobacterial ribosomal proteins with CD4+T cells from IKEPLUS-immunized mice, we identified six additional immunogenic peptides, derived from six different mycobacterial ribosomal proteins. Our results demonstrate that the CD4+ T cell response in IKEPLUS-immunized mice has multiple targets including components of the mycobacterial ribosome. Further studies to more fully define the CD4+ T cell responses to ribosomal targets should help to inform the design of effective vaccines against Mtb.
TGF-β-mediated inhibition of IFN-γ production by Mycobacterium tuberculosis-specific T cells in the infected lung
Authors: Adams Kristin N , Moguche Albanus O , Plumlee Courtney R , Urdahl Kevin B
Published: January 2023

Tuberculosis (TB) is a major global health problem killing over one million people every year. The emergence of multi and extremely-drug resistant (MDR and XDR) strains of Mycobacterium tuberculosis (Mtb) has made treatment difficult and has spurred interest in developing host-directed immunotherapeutics. A critical roadblock is our failure to understand how Mtb escapes eradication despite an apparently robust adaptive immune response. Interferon-γ (IFN-γ) producing CD4 T cells are critical for immunity, yet their abundance in the blood does not correlate with protection to TB, suggesting that their activity may be blocked locally at the primary site of infection in the lung. A leading candidate for IFN-γ suppression in the lung is transforming growth factor-β (TGF-β), a multifunctional cytokine that is capable of inhibiting IFN-γ. TGF-β is produced in excess at sites of active Mtb infection in both murine and human TB. Neutralization of TGF-β improves T-cell responses of active TB patients in vitro, but the extent of its role during in vivo TB is unknown. Here, we generated mixed bone marrow chimeras using WT mice and mice with a T cell-specific conditional deletion of TGF-β receptor II (dLck-Cre:TGFbRIIfl/fl). Mtb-specific TGF-βRII knockout cells outcompeted their WT counterparts and exhibited increased IFN-γ production during Mtb infection. Furthermore, Mtb-infected dLck-Cre:TGFbRIIfl/fl mice had decreased bacterial loads early during infection. Finally, two pharmacological inhibitors of TGF-β signaling lowered the bacterial loads of lungs from Mtb-infected mice both early and late during infection. Targeting TGF-β may pave the way for new host-directed immunotherapeutics for TB.
The tetraspanin CD151 is an activation molecule that characterizes M. tuberculosis- specific effector CD4+ T cells
Authors: Seu Lillian , Sun Jim Jian , Schaaf Kaitlyn Ruth , Duverger Alexandra Emilie , Kutsch Olaf , Goepfert Paul Andres
Published: January 2023

Tetraspanins are a family of membrane proteins regulating cell morphology, motility, fusion, and signaling. CD151 is expressed on T cells that stabilizes the immune synapse during antigen recognition, engages in integrin signaling, and primes T cell activation. We have recently demonstrated that CD151 expression leads to increased proliferation and activation in human CD4+ T cells. Given its important functional role, we hypothesized that CD151 may be elevated on M. tb-specific effector CD4+ T cells. We assessed CD151 on ex vivo PBMCs from PPD+ donors (Purified Protein Derivative from M. tb) stimulated with M. tb H37Rv whole cell lysate. CD151+ frequencies were elevated on IFN-γ+ (52 ± 6% vs. 17 ± 2%, P<0.001) and TNF-α+ (74 ± 6% vs. 17 ± 2%, P<0.0001) CD4+ T cells compared to overall baseline levels. To assess the functionality of CD151 on M. tb-specific CD4+ T cells, we established an ex vivo culture model with M. tb-infected monocyte-derived macrophages (MDMs) in the presence of autologous PPD+ or PPD− donor CD4+ T cells. Upon day 6 of culture, PPD+ donor cultures, proliferating Ki67+ CD4+T cells were elevated in CD151 and CD25, but diminished in CD57, cell surface expression. Additionally, enhanced frequencies of CD151+ Ki67+CD4+T cells were associated with reduced M. tb growth within autologous MDMs during ex vivo culture. In summary, CD151 is a novel T cell activation marker that is elevated on M. tb- specific CD4+ T cells, and is associated with diminished M. tb growth in culture. CD151 could thus be an interesting candidate to supplement the existing TB-TAM diagnostic (T cell activation marker of tuberculosis) – the first to assess the quality of the CD4+ effector T-cell response to M. tb – and may guide future CD4+ T-cell based vaccine strategies.
Kinome analysis of chicken macrophages infected with Salmonella Enteritidis or S. Heidelberg
Authors: He Haiqi , Arsenault Ryan J , Genovese Kenneth J , Kogut Michael H
Published: January 2023

Macrophages are important innate immune cells that play a central role in the first line defense against microbial infection. Although, chicken macrophages are equipped with effective antimicrobial mechanisms that can be readily activated in response to microbial infection, many Salmonella strains are able to survival inside the macrophage. A previous study has shown that S. Enteritidis was better equipped to survive in the chicken macrophages than S. Heidelberg, in which S. Enteritidis, but not S. Heidelberg, was able to suppress the host’s nitric oxide response. The interaction between chicken macrophages and Salmonella as well as intracellular survival of Salmonella in chicken macrophages remains poorly understood. Here, we used a species-specific immunometabolism kinome peptide array to identify pathways that are involved in chicken macrophages’ response to Salmonella infection. The kinome assay was performed on chicken HD-11 macrophages collected at 0.5, 2, and 6 hr post-infection with either S. Heidelberg or S. Enteritidis. We observed significant changes of phosphorylation (p ≤ 0.05) in a large number of peptides during the infection: 390, 449, and 575 peptides for S. Enteritidis and 185, 470, and 442 for S. Heidelberg at 0.5, 2, and 6 hr, respectively. Additionally, a relatively small number of peptides that showed infection-induced phosphorylation changes in opposite directions when comparing the two serovars: 142 peptides at 0.5 hr, 141 at 2 hr, and 82 at 6 hr. Analyzing phosphopeptides, we found that a number of pathways involved in immunity, signal transduction, cellular process, and metabolism were significantly altered, in some case differentially, during the infection with the two serovars.
Inclusion of non-Francisella epitopes promotes expansion of high avidity, antigen-specific CD4+ T cells for improvement of existing Francisella vaccines
Authors: Roberts Lydia M , Crane Deborah D , Wehrly Tara D , Fletcher Joshua R , Jones Bradley D , Bosio Catharine M
Published: January 2023

Francisella tularensis subsp. tularensis strain SchuS4 (Ftt) is a highly virulent bacterial pathogen and the causative agent of tularemia. There are no licensed tularemia vaccines and experimental live vaccine strains (LVS) vary in their protective efficacy and are poorly defined. Development of novel vaccines will require improvement of protection over the existing LVS and preferably incorporation of well-defined epitopes. However, protective epitopes for tularemia have not been identified. Surviving tularemia requires a strong polyfunctional CD4+ T cell response. Due to the importance of the CD4+ T cell response, we hypothesized ectopic expression of an epitope recognized by high avidity CD4+ T cells would enhance existing vaccine efficacy, regardless of the epitope’s origin. gp61 and LLO are well characterized CD4+ T cell epitopes derived from LCMV and Listeria, respectively, and are recognized by C57Bl/6, but not Balb/c mice. Expression of gp61 or LLO during primary infection with LVS or Ftt improved survival of C57Bl/6, but not Balb/c mice, indicating the presence of low numbers of high avidity CD4+ precursor T cells alters survival during Francisella infection. Furthermore, mice vaccinated with LVS gp61 had significantly improved survival during low and high dose secondary Ftt infection and decreased bacterial burdens compared to controls. Together, this demonstrates that improved efficacy of current tularemia vaccine platforms is achieved by properly targeting appropriate antigen-specific cellular responses. Moreover, the elucidation of Francisella epitopes that elicit high-avidity CD4+ T cell responses, specifically in humans, will be required for successful vaccine development.
IFN-γ production by CD4 CD8 double positive cells is essential for expansion of Proteobacteria in the neonatal murine intestine.
Authors: Mirpuri Julie , Thomas Sarah , Yarovinsky Felix
Published: January 2023

Background

Expansion of Proteobacteria is seen in the neonatal intestine in both humans and mice. Proteobacteria are also significantly over-represented in preterm infants that develop Necrotizing Enterocolitis (NEC). Neonatal mice are more susceptible to models of NEC. The mechanism of expansion of Proteobacteria in neonatal mice and susceptibility to NEC remain undefined.

Objective

To identify immune modulators that favor expansion of Proteobacteria in the neonatal murine intestine.

Methods

qRT-PCR of bacterial 16S rRNA gene sequence tags of colonic fecal contents from 1–3 week (wk) old wild type (WT), IFN-γ knock out and Rag deficient mice was performed to identify major bacterial groups. qRT-PCR and ELISA of whole colon were used to profile cytokine expression. Flow-cytometry of lamina propria cells was used to characterize cells in 1-,2- and 3-wk old mice. IFN-γ neutralizing Ab was given IP to 3 day old mice and then similarly examined at 2 wks.

Results

2-wk-old WT mice showed increased expansion of Proteobacteria and increased production of IFN-γ. IFN-γ KO mice had minimal expansion of Proteobacteria in the colon, this was also seen in WT mice blocked with IFN-γ Ab. Flow-cytometry revealed a unique subset of CD4 CD8 double positive cells, only present in neonatal mice, that produce IFN-γ in WT mice. Rag deficient mice did not have this subset of cells and also had minimal colonization by Proteobacteria at 2-wks.

Conclusions

IFN-γ promotes the expansion of Proteobacteria in the neonatal intestine. A unique and transient subset of CD4 CD8 cells, that are absent in Rag deficient mice, appear to be responsible for the increased IFN-γ in neonatal mice. This is a potential target for intervention in the prevention of NEC in preterm infants.
Dysbiosis-induced IL-33 contributes to impaired antiviral immunity in the genital mucosa
Authors: Lee Heung Kyu , Oh Ji Eun
Published: January 2023

Commensal microbiota are considered to play an important role in antiviral immunity. However, the consequence of dysbiosis on antiviral immunity remains unclear. We demonstrate that dysbiosis caused by oral antibiotics directly impairs antiviral immunity following viral infection of the vaginal mucosa. Antibiotic-treated mice succumbed to mucosal herpes simplex virus type 2 infection more rapidly than water-fed mice. The susceptibility of antibiotic-treated mice was attributed to compromised effector T cell responses rather than defects in innate immunity. By screening the factors controlling antiviral immunity, we found that interleukin-33 was secreted from vaginal epithelium after the depletion of commensal microbiota. This cytokine suppresses local antiviral immunity by blocking the migration of effector T cells to the vaginal tissue, thereby inhibiting the production of interferon-γ at local infection sites. These findings provide insight into the mechanisms of homeostasis maintained by commensal bacteria, and reveal a deleterious consequence of dysbiosis in antiviral immune defense.
Microbiota-inducible Innate Immune, Siderophore Binding Protein Lipocalin 2 is Critical for Intestinal Homeostasis
Authors: Singh Vishal , Yeoh Beng San , Chassaing Benoit , Zhang Benyue , Saha Piu , Xiao Xia , Awasthi Deepika , Shashidharamurthy Rangaiah , Dikshit Madhu , Gewirtz Andrew T , Vijay-Kumar Matam
Published: January 2023

Lipocalin 2 (Lcn2) is a multifunctional innate immune protein whose expression closely correlates with extent of intestinal inflammation. However, whether Lcn2 plays a role in the pathogenesis of gut inflammation is unknown. Herein, we investigated the extent to which Lcn2 regulates inflammation and gut bacterial dysbiosis in mouse models of IBD. Lcn2 expression was monitored in murine colitis models and upon microbiota ablation/restoration. WT and Lcn2 knockout (Lcn2KO) mice were analyzed for gut bacterial load, composition by 16S rRNA gene pyrosequencing and, their colitogenic potential by co-housing with Il-10KO mice. Acute (dextran sodium sulfate) and chronic (IL-10R neutralization) colitis was induced in WT and Lcn2KO mice with or without antibiotics. Lcn2 expression was dramatically induced upon inflammation and was dependent upon presence of a gut microbiota and MyD88 signaling. Use of bone-marrow chimeric mice revealed non-immune cells are the major source of circulating Lcn2. Lcn2KO mice exhibited elevated levels of entA-expressing gut bacteria burden and, moreover, a broadly distinct bacterial community relative to WT littermates. Lcn2KO mice developed highly colitogenic T-cells and exhibited exacerbated colitis upon exposure to DSS or neutralization of IL-10. Such exacerbated colitis could be prevented by antibiotic treatment. Moreover, exposure to the microbiota of Lcn2KO mice, via cohousing, resulted in severe colitis in Il-10KO mice. Lcn2 is a bacterially-induced, MyD88-dependent, protein that play an important role in gut homeostasis and a pivotal role upon challenge. Hence, therapeutic manipulation of Lcn2 levels may provide a strategy to help manage diseases driven by alteration of the gut microbiota.
Nlrp6 limits inflamation in mouse colon by regulating microbiota
Authors: Seregin Sergey S , Golovchenko Natasha , Schaf Bryan , Pudlo Nicholas , Mitchell Jonathan , Chen Jiachen , Schloss Patrick , Martens Eric , Eaton Kathryn , Chen Grace Yi
Published: January 2023

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) that involves chronic inflammation involving the colon. We have previously shown that NLRP6 protects against the development of chemically-induced colitis. Here, we demonstrate that NLRP6 is important for suppressing the development of spontaneous colitis in the IL-10−/− mice model of IBD. IL10−/−Nlrp6−/− mice have significantly increased inflammation based on histologic scoring, spleen weight, fecal lipocalin levels, intestinal permeability, infiltration of immune cells, and upregulation of proinflammatory mediators. To determine whether NLRP6 protects against colitis by modulating the microbiome, we generated and recolonized germ-free (GF) Nlrp6−/− mice with the microbiota from WT donors and compared the composition of the microbiome to that of recolonized GF WT mice. We determined that GF WT and Nlrp6−/− mice acquire distinct microbiota upon re-colonization. Importantly, Nlrp6−/− mice have reduced a-diversity and dramatically increased colonization of Akkermansia muciniphila, which was also observed in IL10−/−Nlrp6−/− mice. To determine whether Akkermansia contributes to increased inflammation in IL-10−/− mice, we monocolonized GF IL-10−/− mice with Akkermansia muciniphila and found that these mice developed increased colitis compared to GF IL-10−/− mice gavaged with a control non-colitogenic Bacteroides strain. Altogether these results demonstrate that NLRP6 regulates the colonization of Akkermansia, which in turn, can precipitate colitis in a genetically susceptible host. Our work furthered the insight into the pathogenesis of UC and will be instrumental in the development of therapeutics that reduce pathobionts colonization to treat IBD.
Intestinal microbiota influence postnatal thymic T cell development
Authors: Jain Nitya , Seed Brian
Published: January 2023

The immune system faces unique challenges in the period immediately following birth. Thrust from a sterile fetal environment, it is abruptly exposed to an immense array of foreign antigens, the major burden of which is in the form of the microbiota newly colonizing the gastrointestinal tract. Adaptive immunity at the post-natal stage is largely immature, and the newborn’s immune system relies initially on innate immune effectors for protection against infections. In the early post-natal period, increasing numbers of innate and adaptive lymphocytes mature in the thymus and migrate to populate peripheral lymphoid organs including intestinal mucosal tissue. Intestinal microbiota regulate the maturation of the immune system at mucosal sites and also influence host physiology and function beyond the mucosal tract such as the brain and the pancreas. However, whether and how intestinal microbial colonization influences immune cell development in the thymus remains unknown. Here we show that the thymus of neonatal mice harbor precursor cells with distinct gene expression profiles compared to adult thymocytes that could potentially favor the development of innate-like lymphocytes. There are discrete patterns of distribution of PLZF and RORgt expressing thymic cell subsets and the prevalence of these cells is influenced by microbial colonization status of the neonatal intestine. Finally, we propose that migratory dendritic cells are one mediator of thymic-intestine crosstalk that transmit microbiota-specific signal to influence development of these thymic subsets. Thus, intestinal microbiota regulate yet another fundamental aspect of immune function- the development of lymphocytes itself in the neonatal thymus.
Cutaneous microbiota drive the accumulation of IL-17A-producing T cells within the lungs
Authors: Constantinides Michael G , Ridaura Vanessa K , Belkaid Yasmine
Published: January 2023

The microbiome consists of a diverse array of commensal microorganisms that reside at barrier sites of the human body and promote local immune homeostasis. While recent work has demonstrated that intestinal commensals can influence immunity throughout the body, the distal effects of other microbial communities have not been fully explored. Here we show that application of distinct human commensals to the skin of mice induces the accumulation of IL-17A-producing T cells in the lungs. The induction of these cells occurs in a manner that is partially dependent on both antigen presentation and IL-23 signaling. Response kinetics and adoptive transfer experiments suggest that the T cells migrate from the skin-draining lymph nodes, while transcriptional analysis has identified specific chemokine receptors that may facilitate trafficking to the lungs. Furthermore, in the context of pulmonary infection, cutaneous application of commensals promotes the production of IL-17A and recruitment of neutrophils to the lungs. This work demonstrates that the skin microbiome can enhance pulmonary immunity, but also has the potential to exacerbate inflammation through the production of IL-17A, thus providing a possible mechanism for why children with atopic dermatitis have an increased propensity to develop asthma, a progression commonly referred to as the “atopic march.”
Macrophage-Dependent Regeneration of Pulmonary Epithelia Requires Trefoil Factor 2 for Wnt Expression
Authors: Hung Li-Yin , Sen Debasish , Oniskey Taylor K. , Nieves Wildaliz , Urisman Anatoly , Krummel Matthew F , Herbert DeBroski R
Published: January 2023

Coordination between epithelial and myeloid cell lineages may facilitate tissue repair, but mechanistic evidence is lacking. Independently of Type 2 cytokines (interleukin-4/13), and T, B or ILC populations, lung macrophages promoted epithelial proliferation following injury caused by Nippostrongylus brasiliensis or bleomycin sulfate. Multiple myeloid populations up-regulated Trefoil factor 2 (TFF2) following lung injury and CD11c-driven Tff2 deletion impaired the proliferative expansion of pro-SpC+ distal lung epithelial progenitors. Direct interactions between macrophages and damaged epithelia resulted Wnt production, which accelerated epithelial proliferation, trans-epithelial resistance, and barrier function in a TFF2-dependent manner. In summary, the current study demonstrates that TFF2 is a regenerative cytokine expressed by macrophages to facilitate repair of infectious or non-infectious lung damage.
Interferon regulatory factor 1 (IRF1) expression patterns by respiratory epithelial cells reveal non-redundancy of type I versus type III interferon.
Authors: Novatt Hilary , Renn Lynnsey , Theisen Terence , Massie Tammy , Massie Tristan , Rabin Ronald L
Published: January 2023

Types I and III interferon (IFN) are co-expressed by respiratory epithelial cells (REC) in response to viral infection. In turn, these IFN stimulate neighboring REC to express a set of interferon-stimulated genes (ISG) through shared signaling pathways. Whether types I and III IFN have non-redundant functions in anti-viral defense of respiratory infections is unknown. Because transcription factors dictate cellular phenotype and function, we hypothesized that ISG that are transcription factors (TF-ISG) mediate non-redundant functions of types I or III IFN. We treated BEAS-2B human REC with increasing doses of IFN-β or IFN-λ1 alone or together, and measured expression of TF-ISG and a set of canonical ISG by qRT-PCR and Western blot. Alone, IFN-β and IFN-λ1 each induced expression of canonical ISG and a subset of TF-ISG. Expression of several ISG differed in response to type I versus type III IFN, either in peak levels or kinetic patterns, or both. Uniquely, IRF1 is induced early in response to IFN-β alone, but is poorly expressed in response to IFN-λ1. Western blots also revealed that while IFN-β alone induced early and transient IRF1 expression, it was lower but sustained (through 24h) after IFN-λ1 alone. In contrast to transcript expression, the two IFN together enhanced expression of IRF1 protein greater than either alone, and IRF1 expression was sustained through 24h. The distinct selective and rapid expression of IRF1 transcript and protein in response to IFN-β suggests that this TF-ISG mediates non-redundant qualitative functional responses of REC to types I and III IFN.
Challenge with Staphylococcus aureus induces a lipid response in newborn piglets
Authors: Alvarez Sandy , Bartlett Jennifer , Pollock Nicolette , Vazquez Jessica , McCray Paul , Porter Edith
Published: January 2023

The innate immune system provides the first line of defense against microbes. Epithelial cells recognize pathogen-associated molecular patterns and detection leads to induction of antimicrobial peptides, proinflammatory cytokines, and chemokines. Recent evidence suggests that antimicrobial lipids, including fatty acids and cholesteryl esters, are also important effectors in the innate mucosal defense of the airways. We have previously shown that cholesteryl linoleate and cholesteryl arachidonate are active against Pseudomonas aeruginosa and other bacteria, and that the expression of SOAT1, a key enzyme for cholesteryl ester biosynthesis, is increased in sinus mucosa of patients with chronic rhinosinusitis. The objective of this study was to further elucidate the regulation of antimicrobial lipid production by assessing the gene expression of enzymes involved in lipid biosynthesis in an in vivo pig infection model. Newborn pigs were challenged intratracheally with aerosolized heat-killed Staphylococcus aureus (n = 7) or saline (n = 5) and RNA was extracted from trachea tissues 4 h later. Gene expression of the enzymes SOAT1, FADS2, FASN and ACSL1 was statistically significantly increased (p < 0.05 in One-Way ANOVA) in infected animals. In a second experiment (n = 3 for each group), formalin fixed tissues were subjected to a lipid preservation protocol prior to processing for histology and staining with the lipophilic dye Oil Red O. We observed in the trachea of animals challenged with S. aureus an increased staining with Oil Red O consistent with increased lipid accumulation. These data provide evidence that infection leads to increased lipid biosynthesis further supporting the concept of lipid-mediated innate mucosal defense.
Distinct tissue-specific functions for TSLP and IL-33 in the atopic march
Authors: Han Hongwei , Roan Florence , Johnston Laura , Bryce Paul J. , Ziegler Steven F
Published: January 2023

Atopic dermatitis (AD) often precedes asthma and food allergy, indicating that epicutaneous sensitization to allergens may be important in the induction of allergic responses at other barrier surfaces. Our study demonstrated a central role for IL-33 in this “atopic march.” We showed that mice exposed to antigen in the skin, in the presence of IL-33, developed severe airway inflammation when later challenged in the lung, and developed allergic diarrhea following oral antigen challenge. In addition, IL-33-driven allergic disease occurred in a thymic stromal lymphopoietin (TSLP)-independent manner. In contrast, IL-33 signaling was required for local inflammation following epicutaneous TSLP/OVA sensitization and during challenge in allergic gastrointestinal disease, but was dispensable during challenge in TSLP-mediated airway disease. These data reveal critical, site-specific roles for IL-33 in the “atopic march” that lead from atopic dermatitis to allergic asthma and gastrointestinal allergy.
Tissue-resident monocytes promote the establishment of lung-resident CD8 T cell memory following influenza infection
Authors: Dunbar Paul , Wein Alexander N , McMaster Sean Ryan , Hayward Sarah Louise , Kohlmeier Jacob E
Published: January 2023

Lung tissue resident memory CD8 T cells (TRM) have been shown to be critical in protecting against heterologous influenza virus challenge by limiting early viral replication and promoting more rapid viral clearance. Unlike other mucosal tissues, we have found that environmental signals are insufficient for the establishment of lung TRM, and that pulmonary antigen encounter is necessary for TRM to differentiate in the lung. This indicates a likely role for pulmonary antigen presenting cells (APC’s) in the process of lung TRM development, and thus we investigated the role of lung APC subsets on the establishment of lung CD8 TRM following influenza infection. Although many lung APC subsets contain flu nucleoprotein (FluNP) over the course of infection, most lung APC’s do not contain FluNP once virus has been cleared by day 10 post-infection. In contrast, lung monocytes continue to have high numbers of FluNP-bearing cells through day 15 post infection, the same time we observe the appearance of flu-specific CD8 T cells with a TRM phenotype. As these data suggest a potential role for monocytes as drivers of TRM differentiation, we tested mice deficient for the chemokine receptor CCR2 (CCR2−/−), which have a significant defect in the recruitment of monocytes to the lung. In our murine influenza model these CCR2−/− mice had significantly less flu-specific lung CD8 TRM than WT controls. Notably, this defect was not observed in the flu-specific CD8 memory T cells in the lung vasculature or mediastinal lymph node (MedLN), indicating this effect is specific for tissue-resident memory cells. Thus, these data show that antigen presentation by CCR2+ monocytes have a likely role in the lung in the establishment of lung CD8 TRM.
Releasable RapidSpheres™ enable immunomagnetic purification of highly viable and functional immune cells from complex tissues in less than 30 minutes
Authors: Clarke Samuel , Kokaji Andy I , Kellerman Drew W , Ewen Catherine , Chambers Martina N , Chan Mandy , Woodside Steven M
Published: January 2023

Recent advances in cellular therapy and diagnostics promise dramatic improvements in disease prevention and treatment. This research is driven by scientists studying how cells of the immune system communicate and relies on the success of cell-based assays. However, it remains challenging and time-consuming to purify large numbers of high-quality cells from complex tissues. EasySep™ Release is a fast and easy cell isolation method which utilizes the Releasable RapidSpheres™. This novel magnetic particle technology improves cell purities by reducing contaminants and allows for gentle particle removal to mitigate potential interference in downstream assays. The 29-minute protocol involves first incubating target cells with antibody complexes followed by the Releasable RapidSpheres™. Next, labelled cells are purified using a hand-held magnet. Particle-free purified cells are obtained by applying a mild dissociation reagent and a final magnetic separation. We validated EasySep™ Release by purifying T, B and NK cells from peripheral blood mononuclear cell samples containing 5 to 800 million cells. Cell purities were 99.7 ± 0.1% (n=17) for CD3+ T cells, 99.4 ± 0.4% (n=17) for CD4+ cells, 98.8 ± 0.5% (n=17) for CD8+ T cells, 98.6 ± 0.7% (n=6) for CD19+ cells and 95.6 ± 1.7% (n=6) for CD56+ cells. Isolated cells were confirmed particle-free, viable and functional. We obtained similar high performance using unprocessed leukapheresis samples of up to 5 billion cells, demonstrating excellent scalability and compatibility with more complex samples. Finally, we show how EasySep™ Release can be paired with commercially available antibodies or sequential separations to isolate almost any cell type, including those with a complex phenotype.
New dyes for highly sensitive analysis of cells in immunofluorescence applications
Authors: Richter Anne , Vahldieck Marco , Jansen Nicole , Schiminsky Esther , Siewert Christiane , Krauthäuser Susanne , Dose Christian
Published: January 2023

The potential of flow cytometry and fluorescence light microscopy in the analysis of complex biological systems is still limited by the range of bright fluorochromes. The sensitivity of fluorescent probes in cell staining is usually limited by several factors including i) the ability of the fluorochrome to absorb energy and convert it into fluorescence light, ii) the extent to which the intrinsic autofluorescence of the system interferes with the fluorochrome emission, and iii) for certain applications, the resistance of the fluorochrome to fixation agents.

Here we present a new set of fluorescent dyes, VB515 and VB667, which are detected in the channels of flow cytometers and fluorescence microscopes. We demonstrate that these fluorescent molecules are superior to commercially available alternatives, e.g., FITC, Alexa Fluor® 488, VioBright™ FITC, BD™ Horizon Brilliant™ Blue 515, or Alexa Fluor 647, and can even exceed the fluorescence signal of Allophycocyanine (APC)- and Phycoerythrin (PE)-conjugated antibodies. While offering higher brightness than APC- and PE, VB515 and VB667-conjugated antibodies also provide higher stability towards fixation agents in comparison to their protein counterparts. They are thus ideally suited for applications requiring high sensitivity for the detection of especially low expressed cell markers.

With these new fluorochromes a multicolor panel and a gating strategy has been developed for the flow analysis of regulatory T (Treg) cells derived from PBMC and lysed blood. Treg cells play a crucial role in the suppression of non-specific or excessive immune responses. Therefore, the detailed characterization of these cells and their function is of great interest for basic and translational research.
Novel tissue clearing method for preservation of morphology, fluorescence and epitope integrity permits quantitative cellular analysis in intact organs
Authors: Gerner Michael Y. , Li Weizhe , Germain Ronald N
Published: January 2023

Spatial organization of cells within tissues is critical for most biological processes, such as cellular differentiation, signal relay, and localized function. Our understanding of such events is historically based on section imaging, which discards 3-dimensional (3D) information and is problematic for understanding structure-function relationships of complex tissues and rare events. Although recent innovations in tissue clearing methods have paved way for whole organ microscopy, they suffer from various technique-specific artifacts, such as fluorophore quenching, poor epitope preservation, or morphological distortions. Here we report a new clearing method, Clearing-enhanced 3D (Ce3D), which generates excellent tissue transparency, preserves cellular morphology and protein fluorescence, and retains epitopes for antibody-based labeling. We use Ce3D to demonstrate whole-tissue microscopy of various cell types in lymphoid and non-lymphoid organs. Further, when coupled with highly multiplexed antibody labeling and Histo-Cytometry, Ce3D permits quantitative analysis of phenotypically complex cells in intact tissues. Collectively, this methodology provides a comprehensive strategy for whole-organ quantitative imaging and analysis, paving the way for improved understanding of cellular organization and physiology.

This research was supported by the Intramural Research Program of the NIH, NIAID.
Direct immunomagnetic depletion of RBC without hypotonic lysis or sedimentation.
Authors: Peters Carrie , Lee Taylor N , Woodside Steven M , Thomas Terry E , Eaves Allen C
Published: January 2023

Isolation of specific cell types for functional studies frequently requires red blood cell (RBC) depletion as a first step. However, hypotonic lysis of samples may be deleterious to certain cell types, and RBC removal by hetastarch sedimentation is inconsistent. Furthermore, RBC depletion with agents such as ammonium chloride (NH4Cl) is often ineffective, leaving samples that still contain many RBC. We have developed an immunomagnetic method to deplete RBC that is rapid, can be used with a variety of sample types, and leaves untouched total nucleated cells (TNC) ready for downstream assays.

RBCs were removed from four different types of samples (whole blood, leukopak, buffy coat and bone marrow) either magnetically using the EasySep™ RBC Depletion Reagent or by NH4Cl lysis. To perform magnetic RBC depletion, samples were diluted 1:1, the reagent added, and the sample placed in an EasySep magnet. Magnetically labeled RBC were held in the magnet and TNC were poured or pipetted off. The reagent addition, magnet incubation and removal steps were repeated a second time, and in some cases the magnet incubation and removal steps were repeated a third time with whole blood and buffy coat samples.

RBC contamination of the TNC fraction was <2% for whole blood (n=8), leukopak (n=2), and bone marrow (n=5) samples treated with EasySep™, and 12 ± 10% for buffy coat (n=4) samples treated with EasySep™; in all cases this was equivalent to or significantly lower than the RBC contamination in the same samples treated with NH4Cl. TNC recovery from samples RBC-depleted by either EasySep™ or NH4Cl lysis was similar. RBCs can be immunomagnetically depleted from samples in 6–15 minutes, depending on the sample type, and TNC are immediately ready for downstream assays.
Targeted inhibition of proinflammatory cytokines produced by myeloid cells
Authors: Efimov Grigory , Rozov Fedor , Mokhonov Vladislav , Drutskaya Marina , Kruglov Andrey , Tillib Sergey , Nedospasov Sergei A.
Published: January 2023

Proinflammatory cytokines, in particular IL-6 and TNF, are crucial factors in the development of autoimmune immune response. Their systemic inhibition has been successfully used as a therapy of multiple autoimmune and inflammatory diseases. However, recent data from murine genetic models suggest that the cellular source of proinflammatory cytokines may significantly affect their biological effects. Cytokines derived from particular cellular sources may be pathogenic while the same cytokine produced by other cell may play a protective role. Thus, systemic cytokine inhibition may be far from the optimal therapeutic approach. To independently confirm these findings and to evaluate potentially more effective therapeutic approaches we are developing bispecific antibodies, which may serve as inhibitors of proinfllammatory cytokines targeted to specific cells. Using myeloid-specific TNF inhibitor we have demonstrated that limiting TNF inhibition to macrophages and monocytes results in superior neutralization activity in the TNF-induced hepatotoxicity model. This approach could lower both the required dose and potentially the incidence of side effects. Currently we are developing the similar approach for IL-6. Our bispecific reagents are based on two different VhH antibodies, thus we immunized the lama with recombinant IL-6 and selected high affinity single-domain antibodies by phage display.

Funding: Russian Scientific Fund grant 14-25-00160 (anti-IL6 antibodies), RFBR grant 14-04-01656 (anti-TNF).