bluuebunny/crossref_metadata_2025 huggingface dataset:
Defective innate immune responses lead to hepatic damage in CD154-deficient mice following infection with attenuated Salmonella typhimurium
Authors: Fernandez-Cabezudo Maria J. , Bashir Ghada , Al-Sbiei Ashraf , Cabral-Marques Otavio , al-Ramadi Basel K.
Published: January 2023

In addition to its established role in immunoglobulin isotype switching, CD40–CD154 interaction plays a role in the regulation of multiple innate immune system cell types. Of note, we have shown that CD154-deficient (CD154−/−) mice are hyper-susceptible to intraperitoneal infection by attenuated strains of Salmonella enterica serovar Typhimurium. Compared to their normal counterparts, CD154−/− mice exhibited progressive morbidity during the early phases of disease and succumbed to infection 3–4 weeks after inoculation. Since the liver is a key target organ in Salmonella infections, we carried out a comparative analysis of infection-associated hepatic changes at different time-points. Bacterial loads in the liver were significantly higher in CD154−/− compared to WT mice, and this correlated with progressively worsening hepatic damage and rising levels of liver enzymes. Liver histomorphology revealed focal necrosis early in the infection that was self-limiting in WT mice. In contrast, infected CD154−/− mice developed large and generalized necrosis coupled with the appearance of large venous thrombi. Immunohistochemical analysis showed a retarded inflammatory response affecting recruitment of myeloid cells in the liver of CD154−/− animals. Moreover, neutrophil recruitment into the peritoneal cavity, as well as the activation of the respiratory burst, was defective in CD154−/− mice. Taken together, our findings demonstrate that CD154-deficiency results in a failure to mount an appropriate innate immune response to contain bacterial proliferation during the acute phase of disease. This renders the animals incapable of limiting the extent of histopathological damage in the liver resulting, ultimately, in their demise.
Blocking CXCL1-dependent neutrophil recruitment prevents immune damage and reduces pulmonary bacterial infection after inhalation injury.
Authors: Maile Robert , Dunn Julia L. , Kartchner Laurel B. , Jones Samuel W. , Cairns Bruce A.
Published: January 2023

Smoke inhalation associated with structural fires, wildfires, or explosions leads to lung injury. Clinically relevant animal models are needed to develop therapeutics. We have previously reported that damage associated molecular patterns (DAMPs) and anti-inflammatory cytokines correlate with infectious complications in patients diagnosed with inhalational injury. We describe a novel translational murine model of acute inhalational injury characterized by an accumulation of protein and neutrophils in the bronchoalveolar space, and histological evidence of tissue damage. Mice were anesthetized, a cannula placed in the trachea, and exposed to smoldering plywood smoke three times for two minute intervals. Here we demonstrate that this model recapitulates clinically relevant phenotypes including early release of dsDNA, IL-10, MCP-1, and CXCL1 along with neutrophilia early after injury, accompanied by subsequent susceptibility to opportunistic infection with Pseudomonas aeruginosa. The mouse model, and a re-analysis of patient samples, revealed a late release of the DAMP hyaluronic acid (HA) from the lung. Using nitric oxide synthase deficient (Nos2−/−) mice, we found that Nos2 was required for increases in IL-10, MCP-1, and HA following injury but not release of dsDNA, CXCL1 expression, early neutrophilia, nor susceptibility to opportunistic infection. Depletion of CXCL1 attenuated early neutrophil recruitment, leading to decreased histopathology scores and improved bacterial clearance in this model of smoke inhalation compared to untreated injured mice. These data highlight the potential therapeutic benefit of attenuating neutrophil recruitment in the first 24h after injury in patients.
IL-21 regulates NK cell responses during Mycobacterium tuberculosis infection. Padmaja Paidipally, Deepak Tripathi, Abhinav Van, Rajesh Kumar Radhakrishnan, Ramakrishna Vankayalapati1
Authors: Paidipally Padmaja
Published: January 2023

In the current study, we determined the effects of interleukin (IL)-21 on human natural killer (NK) cells and monocyte responses during Mycobacterium tuberculosis (Mtb) infection. We found that Mtb stimulated CD4+ and NKT cells from healthy individuals with latent tuberculosis infection (LTBI+) are major sources of IL-21. CD4+ cells from tuberculosis patients secreted less IL-21 than did CD4+ cells from healthy LTBI+ individuals. IL-21 had no direct effect on Mtb stimulated monocytes. IL-21 activated NK cells produced IFN-γ, perforin, granzyme B and granulysin; lysed Mtb infected monocytes; and reduced Mtb growth. IL-21 activated NK cells also enhanced IL-1β, IL-18, CCL4/MIP-1β production and reduced IL-10 production by Mtb stimulated monocytes. Recombinant IL-21 inhibited Mtb growth, enhanced IFN-γ, IL-1β, IL-18 and MIP-1β; and reduced IL-10 expression in the lungs of Mtb-infected Rag2 knockout (Rag2 KO) mice. These findings suggest that activated T cells enhance NK cell responses to lyse Mtb infected human monocytes and restrict Mtb growth in monocytes through IL-21 production. IL-21 activated NK cells also enhances the immune response by augmenting IL-1β, IL-18 and MIP-1β production and reducing IL-10 production by monocytes in response to an intracellular pathogen.
ASC phosphorylation at Y146 regulates inflammasome activation and pyroptosis
Authors: McAndrew Christian C. , Napolitano Nina M. , Vompe Alex D. , Wewers Mark D. , Gavrilin Mikhail A.
Published: January 2023

Rationale

Inflammasome assembly and activation is a complex process regulated, in part, by posttranslational modification of inflammasome proteins. ASC is a universal adaptor in virtually all inflammasome platforms and its modification is critical in regulation of inflammasome function. In particular, PTK inhibitor AG126 greatly inhibits inflammasome activation.

Methods

We generated stable THP-1 cells with either overexpression of ASC (YFP-ASC) or knock down of ASC (siRNA and CRISPR) followed by ASC knock in. To test the role of ASC phosphorylation in inflammasome assembly and activity, we mutated the highly conserved tyrosine 146 to Y146A. Cells were stimulated with LPS or live bacteria and inflammasome activity was measured by fluorescent microscopy, ELISA, and immunoblotting.

Results

Upon inflammasome activation ASC is released from the cell together with IL-1β, IL-18 and other inflammasome proteins. Inhibition of select tyrosine kinases severely reduces inflammasome activation, pyroptosis and ASC release. Elimination of the potential phosphorylation site Y146 dramatically changes visible ASC complex formation – from a single round speck to a filamentous structure. Cells with the Y146A ASC mutation show significantly abrogated inflammasome activation as compared to a wild type. Screening of a PTK inhibitor library (Selleckchem) linked several tyrosine kinases to regulating ASC phosphorylation and inflammasome activity.

Conclusion

ASC phosphorylation at Y146 by tyrosine kinases is an important regulator of inflammasome activity and pyroptosis.
Lymphocyte-specific protein tyrosine kinase (LCK) is involved in the aryl hydrocarbon receptor (AHR)-mediated impairment of Immunoglobulin secretion in human primary B cells
Authors: Zhou Jiajun , Henriquz Joseph , Crawford Robert , Kaminski Norbert E.
Published: January 2023

AHR activation by the high affinity ligand, TCDD, is widely established to suppress the immunoglobulin M (IgM) response in virtually every animal species tested, and extensively investigated in various mouse models. In mice, activation of AHR is known to impair B cell to plasma cell differentiation and IgM synthesis. In contrast to mouse B cells, activation of AHR in human primary B cells impairs immunoglobulin secretion in the absence of suppressing IgM synthesis. In recent studies, we have identified the putative involvement of LCK in impaired immunoglobulin secretion by human B cells. LCK is a well-characterized tyrosine kinase that phosphorylates known critical signaling proteins involved in vesicular secretion by T cells. Specifically, phosphorylation of tyrosine residue 505 inhibits the activity of LCK. By contrast, little is known concerning the role of LCK in human primary B cells. For the first time, our studies show that activation of the AHR by TCDD upregulates LCK protein expression, which then leads to an impairment of IgM secretion. Treatment with a LCK specific inhibitor restores IgM secretion by human primary B cells. Additionally, the presence of AHR antagonist reverses the AHR-mediated increase of LCK and the impairment of IgM secretion. We also observe a significant increase in phosphorylation of Tyr-505 LCK with TCDD treatment, indicating that AHR activation increases the level of inhibitory LCK. Taken together, our studies revealed a novel and species-dependent mechanism involving the AHR-mediated impairment of IgM secretion and an increase in total as well as inhibitory LCK in human but not mouse primary B cells.
IL-1 and IL-33 differentially regulate the functional specialization of mucosal Foxp3+ regulatory T cells.
Authors: Alvarez Fernando , Fritz Jorg H. , Piccirillo Ciriaco A.
Published: January 2023

CD4+ regulatory T (TREG) cells are critical mediators of peripheral immune tolerance and homeostasis, and express the forkhead box p3 (Foxp3) transcription factor, the master-regulator driving the programming of the TREG cell suppressive phenotype. TREG cells are abundant at mucosal surfaces, where they acquire tissue-specific adaptations. The biological consequences of these adaptations on the stability of thymic-derived TREG (tTreg) cells remain largely unknown. To determine the signals that drive the fate of TREG cells, we isolated and compared stable from unstable TREG cells using a TREG transfer model where we previously observed the functional reprogramming of Foxp3+ TREG cells into Th1/Th17 effector T cells. We identified the expression of the IL-33 receptor (IL-33R, ST2) on stable tTREG cells and the IL-1 receptor (IL1R1) on unstable exTREG cells undergoing functional reprogramming. We show that both TREG cell populations represent competing subsets in inflammatory conditions. This is further underlined by the fact that the absence of IL1R1 expression (IL1R1−/−) leads to the accumulation of ST2+ TREG cells at mucosal sites in vivo. In two distinct lung infection models, we demonstrate that ST2-expressing TREG cells express GATA3 and resist production of inflammatory cytokines, whereas IL1R1-expressing TREG cells express RORγT and lose Foxp3 expression in vivo. While IL-1 signaling impairs TREG cell suppressive function, ST2 is required for the maintenance of the lineage identity and suppressive function of TREG cells. These observations demonstrate that IL-1 and IL-33 produced during immune challenge exert distinct roles on the functional adaptation of Foxp3+ tTREG cells at mucosal surfaces.
c-Maf and STAT3 are essential but distinct regulators of RORγt+ and follicular regulatory T cells
Authors: Wheaton Joshua D. , Yeh Chen-Hao , Ciofani Maria
Published: January 2023

Foxp3+ regulatory T cells (Tregs) must adapt to function within a wide range of tissue environments and types of immune response. This flexibility is achieved through specialization, whereby context-dependent transcriptional changes integrate unique functionality into general Treg programming. In the most well-studied examples, this involves the co-expression of Foxp3 and another “lineage-defining” transcription factor (TF) which confers expression of specific functional genes. Two such specialized populations are RORγt+ Tregs and Bcl-6+ T follicular regulatory (Tfr) cells, which have important functions in regulating colitis and humoral immune responses, respectively. Although the importance of Treg specialization is now clear, the underlying network of signals and TFs driving this process is largely undefined. Here, we identify the TF c-Maf as an essential regulator of Treg specialization for both RORγt+ Tregs and Tfr cells. Importantly, the role of c-Maf in these subsets is highly specific, because deletion of Maf does not affect general Treg development, generation of other specialized Tregs, or expression of suppressive molecules. In vitro, we show that addition of IL-6 to Treg-promoting conditions is sufficient to drive both c-Maf expression and c-Maf-dependent conversion of naïve T cells to RORγt+ Tregs, suggesting that IL-6 might drive c-Maf in vivo. Surprisingly, although STAT3 – the primary downstream effector of IL-6 – is required for development of both RORγt+ Tregs and Tfr cells, we find that Stat3 deletion in vivo does not ablate c-Maf expression and results in Treg defects distinct from Maf deletion. Therefore, c-Maf and STAT3 are cooperative, but distinct regulators of RORγt+ and follicular Treg development.
A C-reactive protein-based strategy to reduce antibiotic dosing
Authors: Ngwa Donald Neba , Singh Sanjay K. , Gang Toh B. , Agrawal Alok
Published: January 2023

C-reactive protein (CRP) binds to Streptococcus pneumoniae through the phosphocholine groups present in the cell wall and subsequently activates the complement system to kill the pathogen. To escape the attack of complement, pneumococci recruit a complement inhibitory protein, factor H, on their surface. CRP protects mice against pneumococcal infection only when injected within 2 hours after administering pneumococci. We hypothesized that CRP is not protective when injected at later times because, by then, factor H is recruited by pneumococci. In the current study, we evaluated the protective effects of an engineered CRP molecule (E-CRP) which does not bind to factor H in fluid phase but binds to factor H-coated pneumococci. We found that E-CRP, unlike native CRP, protected mice regardless of the timing of administering E-CRP by drastically reducing bacteremia and increasing survival of mice. Next, we established another murine model of pneumococcal infection using the antibiotic clarithromycin. We found that the protective ability of 0.2 mg/dose of antibiotic was equivalent to that of 0.02 mg/dose of antibiotic combined with E-CRP; thus, there was 90% reduction in antibiotic dosing by including E-CRP in the treatment. Our data indicate that administration of E-CRP is therapeutically beneficial to reduce antibiotic dosing to decrease bacteremia and enhance survival. These results have implications for infections with antibiotic-resistant pneumococcal strains and possibly for other bacterial species that use factor H to evade the attack of complement. Additionally, the strategy to include E-CRP in the treatment using antibiotics might resolve the problem of developing antibiotic resistance.
IRF3 inhibits IFNγ-mediated restriction of intracellular pathogens in macrophages independently of type I interferons
Authors: Maciag Karolina , Raychowdhury Raktima , Smith Karen , Coers Jörn , Mumbach Maxwell R. , Schwartz Schraga , Polidoro Rafael B. , Gazzinelli Ricardo , Hacohen Nir
Published: January 2023

Macrophages use an array of innate immune sensors to detect intracellular pathogens and to tailor effective antimicrobial responses. In addition, extrinsic activation with the cytokine interferon gamma (IFNγ) is often required as well to tip the scales of the host-pathogen balance toward pathogen restriction. However, little is known about how host pathogen sensing impacts the anti-microbial IFNγ-activated state. We observed that in the absence of IRF3, a key downstream component of pathogen sensing pathways, IFNγ-primed macrophages more efficiently restricted the intracellular bacterium Legionella pneumophila and the intracellular protozoan parasite Trypanosoma cruzi. This effect was not mediated by Type I interferons, the key cytokines known to be induced by IRF3, or by the sensing adaptors MyD88/TRIF, MAVS, and STING. However, IRF3-deficient macrophages displayed a significantly altered IFNγ-induced gene expression program, with upregulation of microbial restriction factors such as Nos2. Furthermore, Nos2 activity was required for the enhanced IFNγ-mediated restriction of T. cruzi observed in IRF3-deficient macrophages. Finally, we found that IFNγ-primed but not resting macrophages largely excluded the activated form of IRF3 from the nucleus. These data are consistent with a relationship of mutual inhibition between IRF3 and IFNγ-activated programs, possibly as a component of a partially reversible mechanism for modulating the activity of potent innate immune effectors such as Nos2 in the context of intracellular infection.
Cannabinoid receptor 1 blockade attenuates metabolic inflammation and gut microbial dysbiosis during high-fat diet-induced obesity.
Authors: Miranda Kathryn , Becker William J. , Busbee Philip Brandon , Menzel Lorenzo , Dopkins Nicholas , Nagarkatti Prakash S. , Nagarkatti Mitzi
Published: January 2023

The endocannabinoid (eCB) system regulates a variety of physiological processes including inflammation and metabolism. Obesity is characterized by overactivation of the eCB system, chronic low-grade inflammation, metabolic dysfunction, and gut microbial dysbiosis. Blockade of cannabinoid receptor 1 (CB1) ameliorates obesity, however CB1-mediated immune-microbial interactions have not been extensively explored. In the current study, antagonists and genetic ablation of CB1 were used in a mouse model of diet-induced obesity to determine the role of CB1 in modulating inflammation, metabolism, and the gut microbiome. As expected, CB1−/− mice were resistant to high-fat diet (HFD)-induced weight gain and metabolic inflammation was significantly less in CB1−/− mice when compared to wild-type (WT) controls. Lamina propria lymphocytes from CB1−/− mice did not display a Th1 induction after HFD feeding whereas WT mice fed HFD had elevated CD4+T-bet+ cells compared to WT mice fed low-fat diet. Decreased colonic inflammation indicated CB1-dependent alterations in the gut microbiome may contribute to a decreased obese phenotype. Similarly, WT obese mice treated with the CB1 antagonist AM251 experienced weight loss, decreased inflammation, and improvement in metabolic parameters. Interrogation of the gut microbiome by 16S metagenomics revealed CB1-dependent alterations in abundance of bacteria belonging to the Clostridiales order. In addition, functional predictions by PICRUSt analysis revealed decreased abundance of operational taxonomic units belonging to bacterial metabolism and membrane transport pathways. Together these data suggest CB1 regulates metabolism by shaping the immune response and gut microbial community.
Random mutagenesis unveils novel host-pathogen interactions during colonic bacterial infections in immunocompromised hosts
Authors: Liu Yue , Xia Xue , Wan Fengyi
Published: January 2023

Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and the rodent equivalent Citrobacter rodentium (CR) are important causative agents of foodborne diseases. A/E pathogen infections cause severe morbidity and mortality in immunocompromised hosts with low interleukin-22 (IL-22); however, the crucial host-pathogen interactions and the pivotal A/E virulence proteins (effectors) under immunocompromised conditions, remain elusive. We conducted a functional screening of a CR mutant library consisting of ~2,000 mutant strains, generated via transposon-mediated random mutagenesis, and identified several avirulent strains, which abolish CR-induced severe morbidity and mortality in IL-22 knockout (Il22−/−) mice. Recapitulating the attenuated virulence phenotype by the transposon-interrupted mutant strains, genomic deletion of each identified effector in CR dramatically attenuates CR-caused lethality in Il22−/− mice. Interestingly, genetic deletion of each identified effector gene in CR genome exhibits no impact on the colonization and proliferation of CR in the infected animals; however, the deficiencies in these effectors substantially impede the colonic inflammatory response in the CR-infected Il22−/− mice, through distinct cellular and molecular mechanisms. Hence, our findings unveil a set of novel A/E pathogen effectors pathologically relevant to the severe morbidity and mortality in Il22−/− animals, which could provide new strategies to control A/E pathogen infections under the low IL-22 immunocompromised conditions, which are frequently associated with chronic HIV infection, organ transplantation, and other diseases.
IFN-γ deficiency alters splenic compartment cellular composition and Brucella distribution in a murine model of brucellosis
Authors: Dadelahi Alexis S. , Lacey Carolyn A. , Skyberg Jerod A.
Published: January 2023

Despite more than a century of research, brucellosis remains a major zoonotic threat with an estimated 500,000 human cases annually. No licensed vaccine exists to counter Brucella infection, leaving many who contract brucellosis with a chronic and debilitating disease. Immunohistochemistry studies have identified targets of Brucella infection in vivo. However, due to the low detection limit of this technique, high infectious doses of Brucella or immunodeficient mice are employed which likely changes the composition of the infected tissue. We and others observed increased splenic bacterial burden and susceptibility to Brucella infection in mice deficient in IFN-γ. Therefore, to determine if IFN-γ changes splenic composition and cellular distribution of Brucella, we performed live cell sorting on spleens from Brucella- infected wild-type (WT) and IFN-γ−/− mice. Spleens from Brucella-infected IFN-γ−/− mice exhibited a lower proportion of B cells and an increased proportion of neutrophils and macrophages compared to WT mice. Interestingly, 90% of intracellular, viable Brucella from WT spleens during chronic infection was in B cells while the proportion of bacterial burden was similar amongst B cells, macrophages, and neutrophils within IFN-γ−/− spleens. In studies using B-cell deficient (μMT−/−) mice challenged with Brucella, we found significantly decreased bacterial burden in μMT−/− spleens compared to WT mice despite similar levels recovered from the joint between both groups. Collectively, these findings indicate that IFN-γ deficiency alters the distribution of cells within the splenic compartment and that B cells enhance Brucella burden in the spleen.
Peripheral Francisella Tularensis Infection Results In Neural Invasion and Pathologic Inflammation
Authors: Ramos Muniz Mireya G. , Spencer Charles T. , Contreras Ryan , Nunez Guillermo
Published: January 2023

Francisella tularensis infection triggers an overactive inflammatory response by stimulating production of host pro-inflammatory cytokines. Human tularemia, caused by F. tularensis, is a zoonotic disease transmitted by aerosol particles, direct contact, and via arthropod vectors, particularly ticks. With the known peripheral invasion of F. tularensis, and previously published clinical data indicating the presence of our bacterium in cerebral spinal fluid, many questions arise, which include the questions as to which specific neuronal cells are infected. In the periphery, macrophages provide innate immune defense against foreign material, including bacteria. Microglial cells are resident macrophages of the brain and spinal cord and previously have been reported to rapidly respond to pathological changes in the central nervous system, serving a similar function as macrophages. Their rapid activation is an important factor in guarding the neural parenchyma against infectious diseases, inflammation, and neurodegeneration while maintaining and facilitating the return to tissue homeostasis. Therefore, we hypothesized that, having invaded the brain by an unknown mechanism, F. tularensis may be capable of eliciting an immune response following infection of microglial cells. Since macrophage infection leads to elicitation of inflammation, infection of microglia similarly stimulate inflammation leading to neural pathology. Mice were inoculated on the rear flank with fluorescently labeled bacteria. Using fluorescent confocal microscopy, labeled F. tularensis was localized in the brains of infected mice. We analyzed brain homogenates for production of inflammatory cytokines at these sites through Milliplex and qPCR.
Mast Cells Play a Protective Role in Spontaneous Colitis
Authors: Lennon Elizabeth M. , Woodrow Jane S. , Thomason Courtney A. , Borst Luke B. , Edwards Laura E. , Moeser Adam James
Published: January 2023

Mast cells are modulators of inflammation and are activated during inflammatory bowel disease (IBD). Previous work has demonstrated that mast cells are proinflammatory in acute models of chemical colitis. However, the role of mast cells in chronic, spontaneous IBD models remains to be fully defined. We hypothesized that mast cells play a protective role in chronic, spontaneous colitis. We compared colitis and intestinal barrier function in IL10−/− mice to mast cell-deficient, colitis prone mice (double knockout (DKO): IL-10−/− x KitWsh/Wsh). Systemic engraftment of DKO mice with mast cells was performed. Mast cell-deficient DKO mice exhibited more severe colitis compared to mice that lacked IL-10 alone, indicated by increased colitis scores, mucosal hypertrophy, increased intestinal permeability, and spontaneous colonic cytokine production. Reconstitution of DKO mice with mast cells prevented colitis (reduced colitis scores, mucosal hypertrophy, TNF, and intestinal permeability). PCR array analyses demonstrated the influence of mast cells on cytokine expression. Analysis of the microbiota revealed sex-specific differences between IL10 and DKO mice, in particular, the abundance of Akkermansia muciniphila, a mucus degrader associated with colitis. Derivation of DKO mice into a germ-free environment revealed that the microbiota is required for disease development. RNA-seq on colon samples revealed candidate genes for the protective effects of mast cells. These results demonstrate that mast cell deficiency exacerbates disease in a chronic, spontaneous IBD model, indicating that mast cells may have protective functions in IBD, and potentially other chronic inflammatory disorders.
Lymphatic and uterine B cells contribute to maternal immunological tolerance during murine pregnancy
Authors: Genuino Ruth Marian Guzman , Eldi Preethi , Garcia-Valtanen Pablo , Hayball John D. , Diener Kerrilyn R.
Published: January 2023

B cells are best known for their ability to instigate positive immune responses by facilitating immune memory and producing antibodies. However, there are subpopulations of B cells that are capable of negative regulation. Collectively referred to as ‘regulatory B cells’ or ‘Bregs’, these B cell subpopulations have been shown to restore immune response to homeostatic levels in conditions requiring calibration of inflammation such as autoimmunity, cancer, and graft tolerance. Bregs carry out negative regulation by producing anti-inflammatory cytokines such as IL-10 and/or suppressing other immune cells by cognate interactions. In semi-allogeneic pregnancy where establishment of maternal immunological tolerance towards the fetus is the defining condition requisite to pregnancy success, the role of B cells in the local tissues has yet to be elucidated. In this study, kinetics of B cells during BALB/c x C57/BL6 murine pregnancy demonstrate transient increase in IL-10-producing B cells at insemination (0.5 days post-coitus), implantation (5.5 days pc), and delivery day in the para-aortic lymph nodes, as well as a significant increase in the uterus at 5.5 days pc and delivery day relative to the estrus virgin state. Moreover, flow cytometry revealed that these IL-10-producing B cells in the lymph nodes and uterus have elevated expression of PD-L1. In vitro suppression assays show that co-culturing lymphatic and uterine B cells from 5.5 days pc pregnant mice with CD4+ T cells significantly reduced the proliferation of T cells. These results suggest that B cells in the local pregnancy milieu function as regulatory B cells that contribute to the establishment of maternal immune tolerance during pregnancy.
ILC2 activation by leukotrienes: NFAT joins the team
Authors: von Moltke Jakob , O’Leary Claire E. , Barrett Nora , Kanaoka Yoshihide , Austen K. Frank , Locksley Richard M.
Published: January 2023

The initiation and maintenance of type 2 (allergic) immune responses in the lung requires production of the type 2 cytokines interleukin (IL)-4, IL-5, IL-13, and IL-9, which together drive eosinophilia and other hallmarks of type 2 inflammation. Group 2 innate lymphoid cells (ILC2s) and type 2 helper T cells (Th2) are the primary source of these cytokines. In Th2s, T cell receptor (TCR) signaling activates the transcription factors NFAT, AP-1, and NFκB, which cooperate to induce type 2 cytokines. ILC2s lack a TCR and respond instead to locally produced cytokines such as IL-33. While IL-33 induces AP-1 and NFκB, NFAT signaling has not been described in ILC2s and it remains unclear how maximal cytokine production is achieved in vivo. Here, we report the non-redundant NFAT-dependent role of lipid-derived leukotrienes in the activation of lung ILC2s. Using cytokine reporter and leukotriene-deficient mice in innate and adaptive models of type 2 airway inflammation, we find that while loss of leukotriene B4 or cysteinyl leukotriene signaling alone has modest effects, deletion of both pathways markedly diminishes ILC2 activation and eosinophilia. Type 2 responses are similarly attenuated in IL-33- and leukotriene-deficient mice, and optimal ILC2 activation reflects potent synergy between these pathways. We hypothesize that ILC2s integrate multiple signals as a substitute for TCR signaling. These findings therefore expand our understanding of ILC2 regulation and may have important implications for the treatment of airways disease.
Evaluation of activation and homing markers on regulatory T cells using a modular flow cytometry approach on the BD FACSLyric™ flow cytometer
Authors: Tyznik Aaron J. , Kara Nihan , Corselli Mirko , Warner Noel L. , Stall Alan M. , Trotter Joe , Saksena Suraj
Published: January 2023

Advancements in cell analysis capabilities have significantly expanded our understanding of the complexity of immunological systems. As the need for deeper analysis has increased, many laboratories have employed a set of lineage markers to define a cell population, followed by the addition of unique markers specific to their biological question. Utilizing regulatory T cells (Treg) as a model population, we describe the design of an 8-color backbone panel on a 12-Color BD FACSLyric™ flow cytometer that can be supplemented with 4 drop-in markers (colors) to characterize two critical facets of Treg biology: activation and homing. Intelligent panel design enabled identification of human naïve and activated Treg cells utilizing established Treg markers (CD3, CD4, CD25, CD127, FoxP3, CD45RA). CD15s and CD161 were included in the 8-color backbone panel to identify functionally suppressive effector and/or pro-inflammatory cytokine secreting Tregs. Importantly, addition of a 4-color drop-in activation panel (PI16, CD147, CD39 and HLA-DR) or a 4-color drop-in homing panel (CCR4, CCR6, CXCR3 and CD31) did not impact resolution of the backbone Treg population(s), while providing new and interesting insights into Treg biology. We highlight the utility of a modular panel design approach that provides an efficient, scalable, and standardized solution for complex analysis of essentially any cell population of interest.
B- and T-cell Immune Repertoire Characterization using Anchored Multiplex PCR and Next-Generation Sequencing
Authors: Haimes Josh D. , Harrison Thomas D. , Nair Namitha M. , Sims Jennifer , McKittrick Ian , Griffin Laura M. , Johnson Laura , Kudlow Brian A.
Published: January 2023

Introduction

NGS-based analysis of the immune repertoire (IR) is a powerful tool to monitor disease, adaptive immune responses to disease, vaccination and therapeutic interventions. IR characterization by NGS usually requires large primer panels, due to the extensive combinatorial diversity, and a complex system of synthetic controls to account for differential amplification efficiency across segment combinations. Anchored Multiplex PCR (AMP™) uses molecular barcoded (MBC) adapters and gene-specific primers (GSPs), enabling NGS-based immune chain mRNA interrogation from a single side. This eliminates the need for opposing primers that bind within the highly variable V-segment, eliminating clone dropout due to somatic hypermutation. Here, we describe AMP-based NGS assays for IR characterization, Immunoverse™ BCR and TCR, which utilize a minimal set of unidirectional GSPs and MBC adapters that reduce amplification bias.

Methods

The quantitative reproducibility and sensitivity of our assays was validated using mRNA isolated from PBMCs of healthy donors, B-cell chronic lymphocytic leukemia donors and formalin-fixed paraffin-embedded (FFPE) tissue.

Results

Both assays demonstrated high reproducibility between replicates with quantitative clone tracking down to 0.01%. The ability to determine isotype, clonotype and IGHV mutational status in a single assay was demonstrated. Preliminary TCR assay data indicates that CDR3 sequence capture is possible from FFPE tissue with clonotype calling being driven by input quantity, T-cell content, and, to a lesser degree, mRNA quality.

Conclusions

AMP-based NGS with MBC quantification and error-correction is a powerful method to characterize the immune repertoire.
Diagnostic Assessment of Immunological History by High-throughput TCR sequence Analyses.
Authors: Wolf Kyle , Maybruck Julie , DiPaolo Richard J.
Published: January 2023

T cell receptor (TCR) rearrangement results in germ-line editing of a T cell’s TCR loci. During infection, T cells with TCRs specific for the pathogen proliferate. Resulting pathogen-specific memory T cells retain the unique germ-line TCR DNA rearrangements, which we hypothesized may be used as molecular biomarkers of prior pathogen exposure. We performed high-throughput sequencing of the TCRβ repertoires of HLA-A2 transgenic mice pre- and post- inoculation with either the smallpox vaccine or highly-related monkeypox virus (MPXV). We generated a database of >2×106 unique TCRβ clonotypes from samples pre- (naïve) and post-exposure to identify TCRs associated with vaccinated/infected samples vs naïve in 58 individual mice. We computationally identified 315 TCRβ sequences statistically associated with post-vaccinated samples, which we used to develop a diagnostic assay to classify samples as naïve or previously-exposed with 97% accuracy up to 9-months post-vaccination. We validated this assay in an independent cohort of MPXV infected mice. The diagnostic platform was adapted to analyze human TCR sequences to distinguish smallpox vaccinated or non-vaccinated individuals. We identified 239 vaccine-associated TCRβ sequences significantly expanded in 100 smallpox vaccinated individuals. A machine learning algorithm was developed to determine individuals’ vaccination status based on the presence of these 239 TCR sequences. Overall, the diagnostic assay correctly identified 74% of non-vaccinated samples and 85% of vaccinated samples. Thus, the analyses support that computational identification of pathogen-associated TCRs is a highly sensitive and specific method for determining prior exposure to an agent or pathogen.
New technology for development of host-targeted therapeutic vaccine against anthrax
Authors: Yang Huixiao , Yan Yongyong , Wang Haiyan , Chen Yanping , Zheng Zhichao , Kim Hyo , Wu Lihong , Franco Susana , Zeng Mingtao
Published: January 2023

Anthrax is a serious disease caused by Bacillus anthracis, a bacterium that can form spores for long-term survival and secrete anthrax toxins including protective antigen (PA), lethal factor (LF), and edema factor (EF). However, current anthrax postexposure treatments are inadequate at later stages of infection. Previously, our research have shown RNA inhibition (RNAi) on host cells silences anthrax toxin receptors (ATRs): tumor endothelial marker 8 (TEM8), capillary morphogenesis protein 2 (CMG2). This approach was protective against the cytotoxicity of anthrax toxins, indicating that ATR-targeted RNAi can be used as a therapy against anthrax. However, inefficient cytosolic delivery and toxicity of siRNA delivery vehicles limit the use siRNA as therapeutics. In this study, we have developed a detoxified anthrax edema toxin, which consists of PA and nontoxic N-terminal fragment of EF (EFn) conjugated with a peptide nona-D-arginine residues (EFn-9dR) to enable siRNA binding. The detoxified toxin was able to deliver specific siRNA to induce cmg2 gene silencing in different cell lines and C57BL/6 mice, and provide significant protection against anthrax lethal toxin challenge in vitro and in mice. Survived mice from toxin challenge were fully protected against lethal challenge with B. anthracis Sterne spores. The immune protective mechanism is mainly due to the significantly high serum neutralizing antibody response against anthrax toxins in these mice. These results suggest that detoxified anthrax toxin provides a tool for delivery of host-targeted siRNA into anthrax pathogenesis-associated host cells, and this new system could potentially be developed as a lifesaving postexposure therapeutic vaccine against anthrax.
Multi-parameter flow sorting with novel antibody fluorochrome conjugates that facilitate relabeling of targeted epitopes and reuse of fluorescence channels in downstream applications
Authors: Evaristo Cesar , Pankratz Jennifer , Schmachtenberg Sabine , Bergmann Anna , Jansen Nicole , Krauthäuser Susanne , Meiler Svenja , Yu Zhong , Schiminsky Esther , Völkel Andrea , Siewert Christiane , Dose Christian
Published: January 2023

Multi-parameter immunofluorescent labeling is the method of choice for flow sorting of cell populations out of heterogeneous mixtures. However, downstream applications of isolated cells are limited since fluorescence channels and epitopes are blocked by antibody fluorochrome conjugates utilized for the flow sorting experiment.

Here, we describe for the first time new types of antibody fluorochrome conjugates that enable a highly specific multi-parameter cell labeling. Unlike conventional fluorochrome-conjugated antibodies, the introduced conjugates rely on recombinantly engineered antibodies and a conjugation chemistry that facilitates the release of the whole conjugates from the cell surface after the flow sorting step. Thereby, previously blocked epitopes are free for relabeling, which provides maximal flexibility for renewed epitope targeting in downstream applications.

We demonstrate this flexibility in the context of a workflow for the isolation of highly pure human regulatory T cells (Treg). Tregs labeled with conventional CD4-VioBlue, CD25-PE and the new releasable CD127-APC were enriched by flow sorting. Final identification of the target cells was achieved by intranuclear labeling of the transcription factor FoxP3, a low expressed marker which requires a bright fluorochrome and fluorescence channel with high sensitivity for reliable detection. The selective removal of the CD127-APC conjugate facilitated the reuse of the APC-channel and enables maximum sensitivity for the cell sorting and analysis step.

In summary, this new generation of releasable antibody fluorochrome conjugates for multi-parameter cell labeling paves the way for so far impossible workflows in research and clinical applications.
IFN-γ regulates transcription of key molecules to prevent progressive experimental autoimmune encephalomyelitis
Authors: Robinson Rachel R. , Forsthuber Thomas
Published: January 2023

Multiple sclerosis (MS) is the most common disabling neurological condition of young adults. Most patients are initially diagnosed with relapsing-remitting MS. Problematically; 10 years after diagnosis approximately 60% of MS patients will have converted from the relapsing-remitting form of MS to secondary-progressive MS despite treatments. IFN-γ, a well-studied pro-inflammatory cytokine, has long been implicated in the pathogenesis of MS and its preclinical model, experimental autoimmune encephalomyelitis (EAE), and is often suppressed during treatments for MS. However, previous studies in our lab revealed that mice lacking IFN-γ signaling developed severe and progressive EAE due to increased presence of myelin debris and lipid peroxidation in the CNS, the cause of which is yet to be fully elucidated. We hypothesize that IFN-g prevents disease progression in this progressive EAE model by regulating the expression of molecules that promote the resolution of lipid peroxidation and clearance of myelin debris in the CNS during EAE. We identified a number of key molecules associated with lipid peroxidation which are temporally dysregulated in the absence of IFN-gR using qRT-PCR. To discover additional molecules involved in disease progression we have performed comprehensive RNA sequencing on the CNS of IFN-gR−/− vs WT mice through the course of disease. We will present the result of the analysis of these molecules and supporting studies. The results of our studies will contribute to a more thorough understanding of the underlying mechanisms and may lead to new treatments to prevent progression of MS.
RTA 1701 is an orally-bioavailable, potent, and selective RORγt inhibitor that suppresses Th17 differentiation in vitro and is efficacious in mouse models of autoimmune disease.
Authors: Dulubova Irina , Jiang Xin , Trevino Isaac , McCauley Lyndsey , Liu Liping , Hannigan Linda , Reisman Scott A. , Reisman Scott A. , Ferguson Deborah , Visnick Mike , Wigley Christian
Published: January 2023

Retinoic acid-related orphan receptor γt (RORγt) is a transcription factor that directs the differentiation of Th17 cells, a subset of CD4+ helper T cells involved in autoimmune disorders. Once differentiated, Th17 cells secrete interleukin-17A (IL-17A) and other key cytokines implicated in autoimmune and inflammatory disease. Antibodies that target Th17 cells are approved for several autoimmune disorders, validating the important role of Th17 cells in disease. RTA 1701 is a potent and selective RORγt inhibitor in preclinical development for autoimmune disorders. It inhibits the activity of RORγt, as assessed using ligand binding domain and full-length RORγt reporters. Moreover, RTA 1701 is selective for RORγt and does not inhibit the activity of two closely-related receptors, RORα and RORβ. By inhibiting RORγt, RTA 1701 blocks Th17 differentiation of naïve CD4+ T cells and IL-17A production by both Th17 polarized CD4+ T cells and activated peripheral blood mononuclear cells (PBMCs). The expression of other RORγt target genes and Th17 signature genes (eg, IL-17F, IL-21, CCL20) is also reduced following RTA 1701 treatment of Th17-polarized naïve CD4+ T cells, as assessed by qPCR. RTA 1701 is orally bioavailable with excellent pharmacokinetic properties in rodents and monkey. Furthermore, this compound is efficacious in animal models of autoimmune disease, including the collagen-induced arthritis (CIA) mouse model of rheumatoid arthritis and the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Together, these results suggest that RORγt inhibition by RTA 1701 may have therapeutic potential in Th17-associated autoimmune disorders.
IL-12p35 suppresses encephalitis and uveitis in mice
Authors: Choi Jin Kyeong , Yu Cheng-Rong , Mary Mattapallil , Lee Hyunsu , Kang Minkyung , Oladipupo Favour , Caspi Rachel R. , Egwuagu Charles E.
Published: January 2023

Interleukin 35 (IL-35) cytokine is comprised of two subunits, IL-12p35 and Ebi3 and both subunits are thought to be required for the expansion of regulatory B cells (Bregs) that suppress autoimmune diseases. Although use of IL-35 to produce Breg cells has substantial promise for the development of cell therapy against autoimmune and neurodegenerative diseases, some significant challenges have yet to be overcome before it can brought to the clinic. For example, the association of the IL-12p35 and Ebi3 subunits is not strong and readily dissociates, making it difficult to ascertain the effective dose of the bioactive IL-12p35:Ebi3 heterodimer administered or required to ameliorate disease. In this study, we show that IL-12p35 alone has anti-inflammatory properties that recapitulated essential therapeutic functions of the heterodimeric IL-35 cytokine. IL-12p35 induced the expansion of Breg cells in vivo and adoptive transfer of ex-vivo-generated Breg cells using IL-12p35 suppressed inflammation and the development of pathology in mouse models of experimental autoimmune uveitis (EAU) or encephalomyedlitis (EAE). The inflammatory immune cells penetrated the retina, brain and spinal cord in the untreated mice whereas these traits were far more reduced in the IL-12p35-treated mice. In conclusion, the recapitulation of essential immunosuppressive activities of IL-35 by IL-12p35, indicates that IL-12p35 may be utilized for in vivo expansion of Breg cells and autologous Breg cell immunotherapy. These and our unpublished observations suggest that IL-12 single chain proteins may constitute a new generation of therapeutic cytokines that can be exploited to treat autoimmune and neurodegenerative diseases.
The role of CD39-expressing regulatory T cells following treatment with αCD52: an effective therapeutic against central nervous system autoimmunity
Authors: Brass Sara Colpitts , Burgess Joseph N. , Begum-Haque Sakhina , Kasper Lloyd H.
Published: January 2023

Alemtuzumab (αCD52), a novel immunotherapy approved for the treatment of relapsing multiple sclerosis (MS), induces the widescale deletion of lymphocytes, including self-reactive T cells. Using the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), we previously found that following reconstitution of the immune compartment, there was an increased frequency of CD39+Foxp3+ regulatory T cells (Tregs) in the spleen and gut-associated lymphoid tissues of αCD52-treated mice. We hypothesized that the CD39-expressing Tregs that emerge following lymphocyte reconstitution would be suppressive against central nervous system autoimmunity. However, when CD39+ Tregs were purified from αCD52-treated mice and adoptively transferred to naïve recipients, they were unable to reduce the severity of EAE disease. We next used Foxp3/GFP reporter mice treated with αCD52 to compare the suppressive capacity of CD39+Foxp3+ versus CD39+Foxp3− Treg subsets and found that CD39+Foxp3+ Tregs, but not CD39+Foxp3− Tregs, were able to significantly reduce the proliferation of activated naïve T cells in vitro. Finally, we sought to determine if expression of CD39 was required for the therapeutic effects of αCD52 in the EAE model. Preliminary studies using a limited number of CD39 KO mice indicated that treatment with αCD52 was equally effective in both control and KO mice. Thus, our studies have identified potential differences in individual subsets of Tregs emerging after αCD52 treatment and suggest that expression of CD39 is dispensable for the short-term effectiveness of αCD52 in mice. Future studies are required to determine if CD39 expression on Tregs contributes to the long-term suppression of MS disease in patients treated with αCD52.