bluuebunny/crossref_metadata_2025 huggingface dataset:
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) attenuates inflammatory response to pertussis toxin by induction of Tregs and immunosuppressive cytokine production via microRNA modulation
Authors: Al-Ghezi Zinah Zamil , Mehrpouya-Bahrami Pegah , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a halogenated aromatic hydrocarbon, is one of the most toxic compounds known and a wide-spread and persistent environmental contaminant. This chemical is a well-known contaminant in Agent Orange, a herbicide used in the Vietnam War. TCDD was released into the environment in the Seveso disaster. It is usually present in a complex mixture of dioxin-like compounds and is a carcinogen. TCDD has also been shown to act as a potent immune suppressor. In this study, we investigated the effects of TCDD on immune status by examining the splenocytes from mice following i.p. injection with pertussis toxin (PTX). We assessed cytokine levels and Treg numbers in control (PTX+Vehicle) and treated mice (PTX+TCDD). We found that TCDD-treated mice had increased levels of IL-10, while IL-6 decreased. In addition, mice treated with TCDD showed overall decreased levels of CD4+T cells and pro-inflammatory cytokines, IL-17 and IL-6. However, increases in the levels of Tregs (FoxP3+ CD4+T cells) and anti-inflammatory cytokine, IL-10 were observed in TCDD-treated mice. MicroRNA microarray analysis revealed altered miRNA profile in splenic CD4+ T cells following TCDD treatment of mice injected with PTX when compared to controls. Further evidence indicated that TCDD treatment significantly down-regulated several miRNAs such as miR101b, miR141-3p and miR-142-3p in splenic CD4+ T cells that target immunosuppressive cytokine (IL-10 and TGF-β) and transcription factor (FoxP3) genes. Collectively, these results demonstrate that TCDD suppresses the immune response after administration of PTX through modulating miRNA targeting key immune response genes involved in cytokine production and Treg differentiation.
T cell intrinsic IL-1R signaling licenses effector cytokine production by memory CD4 T cells
Authors: Jain Aakanksha , Song Ran , Wakeland Edward K. , Pasare Chandrashekhar
Published: January 2023

Innate cytokines are critical drivers of priming and differentiation of naïve CD4 T cells but their role in memory T cell function is largely undefined. We discovered that while TCR and CD28 ligation are primary requirements for memory T cell reactivation, IL-1 acts as a licensing signal to permit effector cytokine production by pre-committed Th1 (IFNg), Th2 (IL-13, IL-4 and IL-5) and Th17 (IL-17A, IL-17F and IL-22) lineage cells. This licensing function of IL-1 is conserved across effector CD4 T cells generated by diverse pathogenic insults and different routes of infections. We found that Il1r−/− CD4 T cells from spleen as well as small intestine can differentiate in vivo into Th1, Th2 and Th17 lineages at the steady state. However, the same CD4 T cells are unable to secrete effector cytokines following TCR ligation. Our data demonstrates that successful lineage commitment does not translate into productive effector function in the absence of IL-1R signaling. Acute abrogation of IL-1R signaling in vivo also resulted in reduced in situ IL-17A production by primed intestinal Th17 cells. Consistently, reactivation of circulating memory CD4 T cells from healthy human donors with autologous DCs in the presence of hIL-1R antagonist (Anakinra) resulted in significantly diminished IL-17A production. Mechanistically, abrogation of IL-1R signaling during reactivation led to rapid decay of T cell cytokine transcripts. Conversely, the presence of recombinant IL-1b during T cell reactivation rescued destabilized cytokine transcripts in a p38 dependent fashion. Together, these results uncover a novel layer of regulation of memory CD4 T cells and establish a broadly conserved role for IL-1 in memory CD4 T cell effector function.
A novel double edged sword in T cell development and function: Receptor-Interacting Protein Kinase 1 (RIPK1)
Authors: Thiault Nicolas , Chen Angeline , Verstichel Greet , Huysentruyt Jelle , Vandenabeele Peter , Cheroutre Hilde
Published: January 2023

Ripk1 is a key regulator of survival and death. This dual function is controlled by post-translational modifications: ubiquitination of RIPK1 leads to activation and survival whereas its deubiquitination or phosphorylation triggers death. In addition to its involvement in downstream signaling by many death receptors RIPK1 also functions in TCR-induced signaling cascades that either lead to activation-induced death or survival and NFkb activation. The role of Ripk1 in T cells development and functions are still poorly understood mainly because of the perinatal lethality of Ripk1 genomic deletion. To circumvent this problem, we generated T cell specific conditional KO mice by crossed Ripk1fl/fl mice with CD4-Cre. This deletion during development greatly affected the generation of TCRab thymocytes and in particular the agonist selected T cell subsets, including the NKT cells and the TCRab DN T cells. Similarly, TCRab T cells in the periphery were greatly reduced, in particular the NK T cell and DN subsets, whereas as expected TCRgd T cells were not affected. Most remaining TCRab T cells displayed a CD44+ activated phenotype and differentiation to cytotoxic cells, including the CD4 T cells. A similar effect was seen among the mucosal T cells in the gut, where few DN T cells were present and most of the intraepithelial TCRab T cells were cytotoxic CD8 T cells with high levels of Granzyme expression. All CD4 IETs were negative for ThPOK and expressed granzyme similar to their CD8 counterparts.

These observations highlight an important role for RIPK1 in thymic development of agonist selected T cells as well as conventional CD4 and CD8 thymocytes and in the periphery for the functional maturation and survival of lymphoid and tissue resident T cell subsets
Normoxic to hypoxic switch of pre-activated NK cells leads to robust proliferation and enhanced effector function via stabilization of HIF-1α and inhibition of apoptosis
Authors: Kim Jungwon , Lim Seon Ah , Moon Yunwon , Shin Min Hwa , Cassian Yee , Park Hyungsung , Lee Kyung-Mi
Published: January 2023

Although mature NK cells can function within hypoxic lymphatics and tissues, they show impaired anti-tumor functions in ex-vivo hypoxic environments. This raises a question regarding additional cues that may exist to allow full NK cell activation within the hypoxic tumor microenvironment. Here, we provide evidence that pre-activation of NK cells prior to hypoxic exposure is key to overcoming hypoxia-mediated impairment and inducing greater NK cell proliferation with enhanced cytolytic activity. Specifically, exposing pre-activated NK cells to 1.5% of pO2 hypoxia following 7-9 days of normoxic culture allowed HIF-1α stabilization and its target gene expression, leading to specific upregulation of NKp44 activating receptor and increased STAT3 phosphorylation. These hypoxia-exposed NK cells demonstrate concomitant reduction of apoptotic and p21/p53 senescence pathways, resulting in greater cell numbers in culture. Therefore, pre-existing proliferative signals allows HIF-1α-mediated adaptation of NK cells toward highly proliferative and cytolytic phenotypes, which may occur in vivo throughout tumor immunosurveillance.
The NF-kappa B regulator Bcl-3 controls T cell differentiation during challenge with the lymphocytic choriomeningitis virus
Authors: Jaiswal Hemant , Siebenlist Ulrich
Published: January 2023

Bcl-3 is an atypical member of the IκB family. Unlike classical IκBs, Bcl-3 is not degraded in response to activation signals, but instead its expression is often induced and it acts in the nucleus to modulate transcription of NF-κB target genes by associating with p50/NF-κB1 and p52/NF-κB2 homodimers. The latter complexes do not trans-activate by themselves, and association with Bcl-3 may promote or inhibit transcription in a highly gene- and context-dependent manner. Bcl-3 has profound biologic roles. Bcl-3−/− mice exhibit notably impaired adaptive and innate immune responses. Previously, we have shown that Bcl-3 regulates plasticity and pathogenicity of autoimmune CD4 T cells in the T cell transfer-induced colitis model as well as in EAE. However, it is not understood whether and how Bcl-3 may intrinsically regulate differentiation and functional responses of T cells during pathogenic challenges. To investigate for roles of Bcl-3 in primary and secondary responses to a viral pathogen, mice lacking Bcl-3 specifically in T cells (Bcl-3 ΔT) were challenged with acute lymphocytic choriomeningitis virus (LCMV) infection. LCMV antigen-specific CD8 T cell recall responses were notably deficient in Bcl-3 ΔT mice, despite comparable primary CD8 T cell antiviral responses at day 8 post infection. Bcl-3 ΔT mice instead exhibited significantly reduced proportions of LCMV-specific CD8 T cells later, by 6 weeks post infection, with remarkably fewer cells exhibiting TCM phenotypic markers (e.g. CD62L and IL-7Rα). This suggests a crucial role of Bcl-3 in long-term maintenance of antigen-specific CD8 T cells. We will present initial investigations into mechanisms by which Bcl-3 may promote antiviral T cell memory formation.
CD99 facilitates rearrangement of actin cytoskeleton during T cell activation.
Authors: Nam Giri , Choi Eun Young
Published: January 2023

CD99 is an O-glycosylated transmembrane protein expressed on most hematopoietic cells and has been known to play roles in transendothelial migration of leukocytes. Several reports showed that blocking CD99 on T cells rendered the cells unable to infiltrate into inflamed tissue sites. To understand how CD99 affects T cell migration, we examined the dynamics of antigen-specific T cells in the presence or absence of CD99 during allo-response. When we adoptively transferred WT or CD99-deficient T cell into B6 mice followed by intraperitoneal immunization of splenocytes from allogeneic mice, CD99-deficient antigen-specific T cells were rarely detected in the peritoneum, the antigen-existing target site, relative to WT T cells on day 3 post-immunization, consistent with previous studies. Unexpectedly, however, CD99-deficient antigen-specific T cells were detected in the peritoneum with comparable numbers to WT antigen-specific T cells at later time point, day 7. This delayed migration in CD99-deficient T cells was caused by slower activation than CD99-sufficient T cells in the spleen. We also found that actin dynamics and microcluster movement were impaired in CD99-deficient T cells during synapse formation, and these defects were due to insufficient Rac1 activation in the absence of CD99. Thus, these results demonstrate that CD99 is an important mediator of actin cytoskeleton rearrangement for adequate T cell activation and migration.
Enhancing student knowledge and understanding of Immunology using integration of a clinically relevant Immunological condition within postgraduate clinical sciences education
Authors: Sika-Paotonu Dianne
Published: January 2023

Proper delivery of Immunology education is fundamental for obtaining detailed understanding of important cellular processes and mechanisms. The inclusion of undergraduate Immunology teaching content within nursing, midwifery and health related courses however may vary. To support effective teaching and learning at postgraduate level for these students undertaking postgraduate clinical sciences with mixed levels of Immunology education, integration of clinically relevant Immunological course elements and related research teaching examples are needed. The purpose of this work was to successfully incorporate a clinically relevant Immunological example into Immunological course teaching. Acute rheumatic fever (ARF) is an autoimmune condition caused by untreated group A streptococcal (GAS) infection of the upper respiratory tract (and possibly skin). Multiple or severe attacks of ARF can cause permanent cardiac damage known as rheumatic heart disease (RHD). Rates of ARF in New Zealand and Australia are among the highest in the world, particularly amongst Māori and Pacific and Indigenous Aboriginal children. ARF as an autoimmune condition was used as a clinically relevant example to support improved understanding of Immunological course teaching elements. Students demonstrated improved confidence and understanding and memory recall of Immunological concepts in addition to improved in-class engagement and ability to answer questions. These observations suggest that the incorporation of a clinically relevant Immunological teaching example supported postgraduate students in their application of scientific content information and facilitated the in-depth understanding of Immunology within the clinical context.
The incorporation of Self-Directed Learning for English Second Language learners within postgraduate Clinical Health Sciences Immunology education
Authors: Sika-Paotonu Dianne , Tait Carolyn
Published: January 2023

The understanding of Immunological processes and mechanisms within postgraduate Clinical Health Sciences education is essential, particularly when considering pathological disease pathways. Ensuring proper learning is achieved by all students is of the utmost importance for teachers especially when considering English as Second Language (ESL) learners. Contemporary learning and teaching methods are becoming increasingly focused on student centered practices such as Self-Directed Learning (SDL) where a student determines their own learning aims and undertakes methods of their own choosing to achieve and assess their learning outcomes. To support effective learning using SDL, it is necessary students are familiar with SDL strategies. The purpose of this work was to support the adoption of SDL strategies by Nursing, Midwifery and Health ESL learners undertaking postgraduate Clinical Health Sciences Immunology education and identify their impact. Face-to-face Immunology teaching sessions for a postgraduate Pathophysiology course were delivered with the inclusion of SDL strategies to support and facilitate effective learning. These strategies were carefully presented to ensure communication was culturally appropriate for all, especially ESL learners. Positive in-class feedback regarding SDL strategy content was received from all students in addition to favorable formal evaluation comments and student satisfaction scores for the Immunology course content. Student assessment outcomes for ESL learners were encouraging. These finding support the incorporation of SDL learning strategies delivered to ensure cultural appropriateness and support effective learning for postgraduate Immunology education.
Knowledge translation for Cancer Immunotherapy and Immunology that is culturally appropriate and relevant within a Pacific context
Authors: Sika-Paotonu Dianne , Laban Luamanuvao Winnie
Published: January 2023

Pacific peoples living in New Zealand and in the Pacific region remain disproportionately affected by some cancers and have less favourable outcomes when compared with non-Pacific populations. Researchers stand to benefit from research efforts that work collectively and in partnership with Pacific communities. Biomedical research efforts to engage and dialogue with Pacific communities in New Zealand have been limited. The purpose of this work was to support the appropriate delivery and knowledge translation of Cancer Immunotherapy and associated Immunological concepts of direct relevance and importance to Pacific communities. A Pacific Fono (gathering) event was held through the local university that invited current and previous postgraduate Clinical Health Science students who were of Pacific heritage. A portion of this event was presented by a female Pacific Biomedical scientist and dedicated to supporting increased knowledge and awareness of emergent cancer immunotherapeutic based strategies for treating cancer. This event was well attended by current and previous Pacific postgraduate Clinical Health Science students of Pacific heritage many of whom are now employed as health professionals and included current and former health leaders, family members, health researchers, community representatives. Positive verbal and written feedback was received from those in attendance with a request for more regular events be held. The Pacific Fono event was the appropriate means to support knowledge translation of Immunotherapeutic strategies for treating cancer by engaging and communicating with Pacific community members in a culturally appropriate, clear and meaningful way.
Neutrophil responses to acute Mycobacterium tuberculosis infection in non-human primates
Authors: Phuah Jia Yao , Junecko Beth , Mattila Joshua T.
Published: January 2023

In human and animal models of Mycobacterium tuberculosis (Mtb) infection, neutrophil involvement is associated with severe active disease and increased tissue pathology in tuberculosis (TB). Transcriptomic analyses of human and non-human primate peripheral blood has identified distinct transcriptional signatures, especially during early stages of infection, that link neutrophils and active disease but the biology of peripheral blood neutrophils in TB remains poorly understood. This study is aimed at understanding neutrophil behavior during early stages of Mtb infection using the non-human primate model of TB infection. To accomplish this, we examined cytokine production, secretion, and gene transcription in cynomolgus macaque peripheral blood neutrophils early after Mtb infection. We found that peripheral blood neutrophils could secrete a surprising range of cytokines in response to mycobacterial stimulation, and in a longitudinal analyses, cytokine expression was upregulated between 3-5 weeks post Mtb infection. Studies on neutrophil transcriptional responses to Mtb were performed to identify changes induced by mycobacterial antigens. These results suggest that changes in host biology during early disease modify peripheral blood neutrophil responses to mycobacteria.
Balance of IL-21 and type I IFN in the granzyme-dependent innate immune response to Staphylococcus aureus
Authors: Spolski Rosanne , West Erin , Li Peng , Kazemian Majid , Leonard Warren J.
Published: January 2023

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a major complication of viral respiratory infections and immunodeficient states. Here, we investigated the role of interleukin-21 (IL-21) in host response to pulmonary MRSA infection and unexpectedly found that IL-21 acts directly on neutrophils and can promote MRSA clearance. When administered intra-tracheally into wild-type mice, IL-21 potently induced granzymes and augmented host defense, and IL-21-induced killing of MRSA was also observed with peripheral blood neutrophils from healthy donors but not from patients with autosomal dominant hyper-IgE (Jobs) syndrome. Unexpectedly, however, loss of IL-21 signaling, as occurs in Il21r KO mice or WT mice treated with an IL-21R-Fc fusion protein or mice expressing a mutant STAT3 transgene, also resulted in enhanced clearance of MRSA, and this was associated with enhanced expression of interferon-dependent genes. Moreover, IFNb induced granzyme B expression by neutrophils, and a granzyme B inhibitor blocked IFNb-induced MRSA clearance. These results reveal a balance and an interplay between IL-21 and type-I IFN signaling in the granzyme-dependent neutrophil response to MRSA, indicating that manipulating IL-21 signaling may be a strategy for controlling pulmonary bacterial infections.
Addition of apoptotic cells alters immune cell function and reduces ankle swelling in experimental Lyme arthritis
Authors: Hilliard Kinsey A. , Brown Charles R.
Published: January 2023

Experimental Lyme arthritis is induced by the infection of C3H mice with the spirochete, Borrelia burgdorferi, (Bb), and is a valuable model to study the regulation of inflammation during bacterial infection. We are interested in understanding the role that apoptotic cells (AC) play in inflammation resolution during murine Lyme arthritis. We show the number of AC within infected ankle joints significantly increases during arthritis resolution. Additionally, injection of AC into the tibio-tarsal joints of Bb infected mice reduces ankle swelling compared to control animals. In vitro studies using bone marrow derived macrophages (BMDM) co-cultured with Bb demonstrates the addition of AC decreases TNFα and KC production, while increasing IL-10 and PGE2 levels. The presence of AC in BMDM and also bone marrow neutrophils (BMN) cultures, decreases Bb phagocytosis, and also decreases BMN migration to LTB4. To further explore this mechanism, we measured expression of PPAR-γ (peroxisome proliferator-activated receptor gamma), which is known to regulate cytokine expression. Addition of AC to BMDM/Bb co-cultures increased PPAR-γ transcription in vitro, and also within infected ankle joints compared to mock treated mice in vivo. We also found AC themselves can produce PGE2, a bioactive lipid which activates PPAR-γ, setting up a positive feedback loop for the production PGE2. These results suggest AC can alter macrophage and neutrophil effector functions and may play a role in downregulating inflammatory responses, perhaps via activation of PPAR-γ. In addition, injection of AC into infected joints may limit inflammatory responses or induce their resolution.
Interferon-Independent Protection by Interferon Regulatory Factor 3
Authors: Gyorke Clare Elise , Nagarajan Uma
Published: January 2023

BACKGROUND

Independent type I/III IFN regulation, interferon-regulatory factor 3 (IRF3) can mediate cell death and suppress proliferation during stress. Novel ‘suicide necrosis’ centers around IRF3 and is one protective mechanism against intracellular pathogens. Uterine epithelial cells infected with obligate intracellular Chlamydia trachomatis are typically shed 3–5 days post-infection (dpi) by unknown mechanism(s) and exhibit new growth. We have previously shown IRF3-deficient mice exhibit decreased epithelial shedding and 10-fold greater bacterial burdens during early infection. We hypothesize that IRF3-mediated cell death restricts chlamydial infection in the genital mucosa, providing a potentially novel mechanism to reduce pathogen load.

METHODS

Progesterone-treated C57BL/6J and IRF3 KO mice were infected vaginally with 3e5 IFUs of C. muridarum strain “Nigg” and histologically assessed 3 and 4 dpi for chlamydia inclusions, cell death and proliferation using proliferation marker PCNA. Cell death was assessed in vivo with PI delivery by NSET device 10 minutes before sacrifice 4 dpi.

RESULTS/CONCL

3 dpi, uterine epithelia in WT and IRF3 KO mice remain intact with no epithelial shedding. WT show a single layer of infected epithelial cells, while IRF3 KO have several layers of infected cells, suggesting the next layer(s) of epithelial cells are generated before the first is shed. 4 dpi we see dramatic increases in epithelial shedding in WT mice when stained in vivo with PI. In contrast, IRF3 KO epithelia show relatively few PI-stained cells. We conclude that IRF3 is playing a significant and protective role during C. muridarum infection by facilitating cell death and limiting cellular proliferation of epithelial cells.
CXCL-1/KC collaborates with CXCL-2/MIP2 to regulate granulopoiesis and recruitment in pneumococcal pneumonia
Authors: Paudel Sagar , Baral Pankaj , Bergeron Scott R. , Ghimire Laxman , Jin Liliang , DeCorte Joseph A. , Le John T. , Cai Shanshan , Jeyaseelan Samithamby
Published: January 2023

Streptococcus pneumoniae remains as a leading cause of community-acquired pneumonia in the U.S. and world. Leukocytes (neutrophils) recruitment to the lungs is critical step in the host defense against pneumococcal infections. CXCL-1/KC is sufficient to induce neutrophil influx in numerous bacterial lung infections, but precise mechanism of how it regulates recruitment to the lungs remains unexplored. Here, we used a mouse model of pneumococcal pneumonia to investigate the role of CXCL-1 in neutrophil granulopoiesis, recruitment, and host defense. CXCL-1 knockout and their wild type controls were infected intratracheally with S. pneumoniae. Survival, leukocyte influx, cytokines/chemokines in bronchoalveolar lavage fluid, and bacterial burdens in lungs and other organs were quantified. Flow cytometry was performed to study different stages of neutrophil granulopoiesis according to their levels of c-Kit and Ly6G expression. Recombinant CXCL-2/MIP2 was administered to rescue neutrophil recruitment in CXCL-1 knockout mice. We here demonstrate that CXCL-1 modulates rapid neutrophil influx, restricts bacterial burden in lungs, regulates production of cytokines/chemokines, and improves host survival. Flow cytometry analysis revealed CXCL-1 is involved in efficient amplification of early neutrophil precursor and release of mature neutrophils from bone marrow. Furthermore, CXCL-2 treatment post-pneumonia corrected the impairment in host defense through increased neutrophil influx in CXCL -1 knockout mice. These results suggest CXCL-1 mediates granulopoiesis and recruitment of neutrophil, and modulation of its activity presents a promising avenue for development of therapeutics against pneumococcal pneumonia.
Investigating the antibacterial effect of Sca1+ lung mesenchymal stem cells on multidrug resistant bacterial species
Authors: Rangasamy Tirumalai , Paudel Sagar , Ghimire Laxman , Jin Liliang , Bergeron Scott R. , Jeyaseelan Samithamby
Published: January 2023

Rationale

Although bone marrow-derived mesenchymal stem cells have been extensively studied in host protection against bacterial pneumonia, the role of lung stem/mesenchymal stem cells in pneumonia caused by the multidrug-resistant bacteria remains elusive.

Methods

We have compared the extracellular (using 10 MOI) and intracellular (using 1 MOI) killing effects of stem cell antigen 1+ lung mesenchymal stem cells (Sca1+ LMSC), bone marrow-derived neutrophils (BMDN), and bone marrow-derived macrophages (BMDM) by infecting them with Gram positive methicillin-resistant Staphylococcus aureus (MRSA) and Gram negative carbapenem-resistant Klebsiella pneumoniae (CRKP) at different time points. We evaluated the extracellular and intracellular killing at each time point by plating them on Tryptic soy agar and MacConkey agar plates, respectively. We also have examined expression of inflammasomes or inflammasome-associated proteins in MRSA (20 MOI for 6 h) infected LMSC using Western blotting.

Results

Sca1+ LMSC is potent in killing (extracellular) both MRSA and CRKP at 3 h post-infection. However, BMDN did not show significant extracellular killing on MRSA and CRKP at all time points examined. Interestingly, Sca1+ LMSC showed a remarkably increased intracellular killing of MRSA than BMDM. Western blotting revealed the increased level of inflammasome and inflammasome-associated proteins (NLRP3, NLRP6 and ASC) in MRSA infected LMSC.

Conclusions

These findings have solidified the potential of LMSC-based therapies for pneumonia caused by MRSA and CRKP. However, in vivo (animal) studies are required to determine the effectiveness of LMSC in the clearance of these bacteria and modulation of innate immune responses in the host.
CXCL5 suppresses IL-17A mediated immunity during MRSA-induced pneumonia
Authors: Ghimire Laxman , Bergeron Scott R. , Paudel Sagar , Jeyaseelan Samithamby
Published: January 2023

Lipopolysaccharides induced CXC-chemokine (LIX or CXCL5) is a potent chemoattractant for neutrophil recruitment to the lung during acute bacterial infections. However, its role in MRSA-induced pneumonia has not been established. We used C57BL/6 (wild-type) and CXCL5 knockout (KO) mice to induce pneumonia by intratracheal inoculation of 5×107 colony forming units (CFUs) of MRSA per mouse. Mice were sacrificed at 12 and 24 hours post-infection to enumerate bacterial burden, determine cellular recruitment, and perform cytokine analysis. Flow cytometric analysis of lungs cells was performed to determine the source of IL-17A. Unlike their wild-type counterparts, KO mice were protected from MRSA-induced pneumonia having significantly higher survival and less bacterial burden in lung and BALF. Total leukocytes and neutrophil recruitment into the airspaces was significantly higher in KO mice. However, proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α were significantly higher in WT mice compared to that of KO mice. In contrast, IL-17A was significantly higher in KO mice. Blocking of IL-17A in the KO mice increased the bacterial burden in the lungs and BALF. In addition, pre-treatment of neutrophils with recombinant IL-17A significantly enhanced ROS production and killing ability of neutrophils in response to MRSA infection. Neutrophils and γδ-T cells were major sources of IL-17A during pulmonary MRSA infection. Together, our findings demonstrate that CXCL5 negatively regulates γδ-T cells and neutrophils driven IL-17A-mediated immunity during MRSA-induced pneumonia and blocking CXCL5 could be a potential therapeutic approach to treat MRSA-infected pneumonic patients.
The oxidized phospholipid oxPAPC ameliorates septic shock by targeting the non-canonical inflammasome in macrophages
Authors: CHU LAN H. , Indramohan Mohanalaxmi , Ratsimandresy Rojo A. , Gangopadhyay Ạnu , Morris Emily P. , Monack Denise M. , Dorfleutner Andrea , Stehlik Christian
Published: January 2023

Inflammasomes are signaling complexes that link the recognition of pathogen and danger associated molecular patterns (PAMPs and DAMPs) by cytosolic pattern-recognition receptors (PPRs) to the activation of Caspase-1, leading to the release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18, and the induction of pyroptosis. In addition to the canonical inflammasome activation by Caspase-1, other inflammatory caspases including murine Caspase-11 and its human orthologs Caspase-4 and Caspase-5 participate in the non-canonical pathway in response to intracellular LPS and Gram negative bacteria escaping the phagosomes, resulting in pyroptotic cell death as well as NLRP3 inflammasome-dependent cytokine release. Contrary to the highly regulated multiprotein platform required for Caspase-1 activation in the canonical inflammasomes, non-canonical inflammatory caspases are directly activated by binding to LPS thus simultaneously act as innate sensors and effectors. It is still largely unknown how the activity of these caspases is regulated. Here we identified the oxidized phospholipid 1-Palmitoyl-2-arachidonoyl-sn- glycero-3-phosphorylcholine (oxPAPC) as a negative regulator of the non-canonical inflammasome in macrophages. In addition to its previously reported TLR4 antagonistic role, oxPAPC directly binds to Caspase-4 and Caspase-11, competes with LPS binding and consequently, inhibits LPS-induced pyroptosis, IL-1β release and septic shock in vivo. Therefore, oxPAPC and its derivatives could provide a basis for novel therapies targeting non-canonical inflammasomes during Gram-negative bacterial sepsis.
TCF/LEF family transcription factors in peripheral Treg homeostasis
Authors: Fu Wenxian , Yang Bi-Huei , Yuan Xiaomei , Dong Yi
Published: January 2023

CD4+Foxp3+ regulatory T cells (Tregs) are key players in preventing lethal autoimmunity and excessive inflammation. However, the homeostatic dynamics of Tregs in peripheral lymphoid and non-lymphoid tissues remains poorly defined. We here report that two TCF/LEF family transcription factors, LEF1 and TCF1, play critical roles in this process. Our studies led to a revised “three-stage” model of Treg peripheral differentiation, delineated by the gradient expressions of LEF1 and TCF1. We found that LEF1− TCF1− subset was bona fide effector Tregs and exhibited a core gene signature shared by various tissue Tregs. Transcriptome and conditional knockout analyses revealed that TCF1 and LEF1 may each control segregated clusters of Treg genes and regulate Treg differentiation in a “division of labor” manner. Together, these data provide novel insights into transcriptional regulation of Treg homeostasis and function.
The TCR/ITK signaling pathway regulates the counterbalance of effector and regulatory T cell development
Authors: Huang Weishan , August Avery
Published: January 2023

The mammalian immune system has evolved both effector and regulatory immune axes, and T cells are the key players in both arms. The immune system sorts, responds to and clears invading pathogens through pro-inflammatory activity, which can cause damage to the affected tissues, and eventually have to be resolved to allow tissue remodeling and resume physiological homeostasis. The balance between pro-inflammatory and suppressive immune functions are critical for host health and survival. The T cell antigen receptor (TCR) signals through the downstream non-receptor interleukin-2 inducible T cell kinase (ITK), which is a key signaling modulator of T cell development, differentiation, activation and function. We find that in the face of different inflammatory and nutritional conditions, the TCR/ITK signaling pathway fine-tunes the balance between effector and regulatory T cell development, such as the ratio between Th17/Treg and Th1/Tr1 cells. This balance is regulated by the TCR/ITK signaling via differential downstream signals and metabolic programs. While ITK is not required for T cell activation, it functions as a rheostat of the TCR to regulate signal strength in response to extracellular antigens, and may be a target to allow therapeutically manipulating the balance between effector and regulatory immunity, dependent on the disease conditions.
Vagotomy decreases splenocytes and MHCII+/CD11B+ cells in the spleen
Authors: Nazarali Rizwan , Shapiro Lee A. , Mukherjee Sanjib
Published: January 2023

The Vagus Nerve is a major component, in conjunction with the spleen, in modulating the systemic anti-inflammatory response.

Adrenergic fibers of the splenic nerve innervate the spleen, where they stimulate a distinct population of memory T cells. These memory T cells subsequently release ACh, thereby reducing macrophage activation and inhibiting TNFα. Therefore this pathway, as it relates to the spleen, is a two-neuron circuit: initiating first in the dorsal motor nucleus of the Vagus, subsequently synapsing at the celiac ganglion, and finally synapsing on the memory T cells.

There are additional lines of evidence demonstrating cholinergic modulation of immune cells in the spleen. For example emerging evidence supports a regulating role of the vagus nerve in mobilizing marginal zone B cells and their subsequent secretion of antibodies, heralding an innate immune response. However, a direct link between splenic B cell number and the vagus nerve has not been previously established. Therefore, we assessed the effects of a left unilateral vagotomy on total splenocytes and MHCII+/CD11+ B cells in the spleen 3 days after vagotomy.

Results

The results indicate that at 3 days after left unilateral vagotomy, there is a significant decrease in the overall number of splenocytes. The results also show that this vagotomy results in a trend toward a significant reduction in MHCII+/CD11+ B cells in the spleen.

Conclusions

Our conclusions based upon these results show that there is a direct influence of left vagus nerve innervation on the number of splenic B cells and that left vagotomy results in an overall decrease in either the proliferation of splenocytes in general including the B-cells, or in cell death of splenocytes including the B cells.
Cytokine regulation in human CD4 T cells by the Aryl Hydrocarbon Receptor and GPR68
Authors: McAleer Jeremy P. , Fan Jun , Roar Bryanna , Primerano Donald A. , Denvir James
Published: January 2023

Th17 cells contribute to host defense on mucosal surfaces but also provoke autoimmune diseases when directed against self-antigens. Identifying therapeutic targets that regulate Th17 cell differentiation and/or cytokine production has considerable value. Here, we study the aryl hydrocarbon receptor (AhR)-dependent transcriptome in human CD4 T cells treated with Th17-inducing cytokines. We show that the AhR reciprocally regulates IL-17 and IL-22 production in human CD4 T cells. Global gene expression analysis revealed that AhR ligation decreased IL21 expression, correlating with delayed upregulation of RORC during culture with Th17-inducing cytokines. We further show that expression of GPR15, GPR55 and GPR68 positively correlates with IL-22 production in the presence of the AhR agonist FICZ. Activation of GPR68 with the lorazepam-derivative ogerin resulted in suppression of IL-22 and IL-10 secretion by T cells, with no effect on IL-17. These data reveal a novel feedback inhibitory pathway on IL-22 and IL-10 production in the presence of AhR agonists.
Dynamic changes in Mycobacterium tuberculosis-induced granulomas: developing new tools
Authors: Marcus Sarah Ann , Herbath Melinda , Fabry Zsuzsanna , Sandor Matyas
Published: January 2023

Continuous cell replacement has been demonstrated in a transplantation model of BCG infected liver, however, the Mtb lung granuloma has yet to be studied as lung tissue is not amenable to granuloma transplantation. To demonstrate the plasticity of Mtb lung granulomas, we treated mice 6 weeks post infection with chemokine inhibitors for 7 days. Treated lungs showed a strong, significant reduction in granuloma size, which suggests continuous cell replacement and indicates that Mtb granulomas are actively maintained during infection.

To examine cell fate in more detail, we utilized STOCK Tg(CAG-KikGR)33Hadj/J mice to photo-tag lung granulomas in situ. Following exposure to 405 nm light KikGR Red (583/593 nm) photoconverted lung regions or cells could be differentiated from unexposed Green (507/517 nm) systemic cells. These mice were aerosol infected with 200 CFU Mtb H37Rv. Portions of infected lungs were photoconverted 4 wpi, harvested up to 7 days post-exposure, and examined, along with mediastinal lymph nodes, by confocal microscopy.

Areas of photoconversion were successfully identified in infected lungs. F4/80+ macrophages and CD4+ T-cells were stained and screened for red versus green fluorescence. Unconverted cells of both types were found, demonstrating the utility of this technique for detecting influx into the granuloma. Efflux of cells from the granuloma was also confirmed by the detection of photoconverted cells within the unconverted MLNs. While further optimization is necessary to use this model for quantitative analysis, these results implicate continuous cell recruitment into granulomatous lesions. Elucidating cell traffic to granulomas with this technique can lead to new therapies for many granulomatous diseases.
Dissecting the pathogenic versus protective roles of IFN-γ and IL-17 in staphylococcal toxic shock syndrome and pneumonia using gene targeted HLA-DR3 transgenic mice
Authors: Rajagopalan Govindarajan , Krogman Ashton , Chowdhary Vaidehi
Published: January 2023

The classical, adaptive T cell response to infectious agents is delayed and is antigen-specific. However, the superantigen (SAg) family of exotoxins, which are readily produced in vivo during Staphylococcus aureus infections, cause an immediate and non-specific activation of T cells, resulting in systemic elevation in many cytokines including IFN-γ and IL-17. While both IFN-γ and IL-17 are pro-inflammatory, they play distinct roles in mediating tissue injury and in immunity to infections. Therefore, to precisely delineate the roles of IFN-γ and IL-17 in immunopathology and immunity to diseases caused by S. aureus and its SAg, we first assessed the expression profiles of IFN-γ, IL-17 family members and their receptors in the spleens of HLA-DR3 transgenic mice challenged with a purified SAg using Illumina microarrays. The expression of ifng gene in spleens of HLA-DR3 transgenic mice was significantly higher than Il17a, Il17d and IL17f at all time-points tested (3, 6, 9, 24, 48 and 72 hrs, 3–4 mice per time point). Serum levels of these cytokines correlated with the gene expression data. Subsequently, HLA-DR3, HLA-DR3.IFN-γ−/− and HLA-DR3.IL-17−/− mice were generated and challenged with purified SAg or infected intratracheally with a SAg-producing strain of S. aureus. While both HLA-DR3 (mortality - 11/19) and HLA-DR3.IL-17−/− (mortality - 15/21) mice succumbed to SAg-induced toxic shock syndrome as well as pneumonia, HLA-DR.IFN-γ−/− mice (mortality - 1/11) were significantly protected (p<0.01) in both disease models. Bacterial loads were not different. In conclusion, our novel study using gene targeted humanized mice reveals that while both Th1 and Th17 cells are robustly activated by SAg, Th1 cells and IFN-γ play a lethal role.
A blood-based transcriptional signature in a novel murine tuberculosis model predicts risk of human tuberculosis progression.
Authors: Plumlee Courtney R. , Gern Benjamin H. , Duffy Fergal J. , Delahaye Jared , Rustad Tige R. , Aitchison John D. , Sherman David R. , Zak Daniel E. , Gerner Michael Y. , Urdahl Kevin B.
Published: January 2023

Tuberculosis (TB) is a highly heterogeneous human disease that develops in a subset of individuals who inhale Mycobacterium tuberculosis (Mtb). Using advanced machine-learning algorithms, we discovered a blood transcriptional signature that identifies Mtb-exposed individuals in the process of progressing to active TB up to 18 months before they exhibit clinical symptoms. This signature may help prevent TB disease by identifying individuals who could benefit from early intervention. However, it fails to identify ~30% of progressors and does not reveal insights into the diverse outcome-governing pathways within the infected lung that underlie TB progression. To develop a tractable system that enables blood-based signatures to be linked to mechanistic pathways within the lung, we pioneered a novel murine TB model that recapitulates key aspects of human Mtb infection. We find that mice infected with a physiologic, ultra-low dose (i.e., 1–3 CFUs) of aerosolized Mtb exhibit a broad range of outcomes, with bacterial burdens ranging from less than 10 to ~106 CFUs within individual lungs. Mice that contain Mtb exhibit well-circumscribed granulomatous structures that share many features with human Mtb granulomas. In addition, we have identified a blood transcriptional signature that distinguishes “controller” and “progressor” mice. Remarkably, this mouse-derived signature is equally as effective as our previously identified human-derived signature at predicting TB risk in humans, confirming the model’s relevance to human disease. This finding enables us to use the tools of the tractable mouse system to address questions central to TB pathogenesis in a clinically relevant model.
Commensal Neisseria Modulates Host IL-6 to Promote Oral Colonization
Authors: Powell Daniel A. , Ma Mancheong , So Magdalene , Frelinger Jeffrey A.
Published: January 2023

Neisseria musculi, isolated from the oral cavity of wild caught mice, weakly (50%) colonizes C57BL/6J (B6) mice but readily colonizes CAST/EiJ (CAST) mice. In vitro stimulation of B6 or CAST splenocytes with WT Neisseria or E. coli LPS showed that CAST mice had a blunted inflammatory response producing significantly lower levels of IL-6 than B6 mice. The use of specific genetic knockouts highlighted a need for an intact innate immune system to prevent colonization. B6-RAG-1−/− were colonized at a similar rate to WT B6 while B6-MyD88−/− and TLR4−/− mice were readily colonized like CAST (100%). In vitro stimulation of B6 BMDM or splenocytes with WT Neisseria yielded only low levels of IL-6. A series of Neisseria mutants in the Type IV Pilus (TFP), a key component of bacterial-host interaction, failed to colonize susceptible CAST mice and showed increased levels of IL-6 compared to WT Neisseria stimulation, indicating that the TFP plays a role in down regulating host IL-6 production. Surprisingly, UV-inactivated Neisseria induced high levels of IL-6 regardless of TFP status supporting that this is an active bacterial process and the presence of TFP alone was unable to down regulate IL-6 production. Consistent with a critical role for IL-6 is preventing colonization, mice deficient for the IL-6 receptor were colonized at a rate of 100% indicating host IL-6 signaling plays a critical role in determining host colonization susceptibility.
Circulating memory is sufficient for protection against Chlamydia muridarum in the murine female reproductive tract
Authors: Labuda Jasmine C. , Pham Oanh , McSorley Stephen J.
Published: January 2023

Chlamydia trachomatis is the most prevalent bacterial STI worldwide and yet no vaccine currently exists to protect the public from this reproductive tract pathogen. Recent studies show that Tissue Resident Memory T cells (Trm) are essential for vaccine-induced protection against Chlamydia trachomatis in mice. Moreover, Trm have been implicated in protective immunity to other reproductive tract pathogens including Herpes Simplex Virus. Here, we examine the requirement of Trm T cells in protection against the murine pathogen Chlamydia muridarum (Cm), commonly used to model human Chlamydia infection. Using parabiosis to test whether circulating memory from mice immune to Cm infection can protect naïve mice from reproductive tract challenge with Cm, we found that naïve mice receiving circulating immunity were fully protected from Cm challenge. Ongoing studies with MHC class-II tetramers will examine the phenotype of protective circulating Chlamydia-specific CD4 memory T cells and whether migration to the reproductive tract is a requirement for protection against secondary challenge.
MicroRNA-10a regulates intestinal mucosal dendritic cell development and function by targeting CD11c
Authors: Huang Xiangsheng , Chen Feidi , Xue Xiaochang , Chen Liang , Yang Wenjing , Xu Leiqi , Bilotta Anthony James , Yao Suxia , Cong Yingzi
Published: January 2023

We have previously demonstrated that microRNA-10a (miR-10a), highly expressed in intestine DC and epithelial cells, and negatively regulated by microbiota, regulated T cell function and pathogenesis of inflammatory bowel diseases by targeting IL-12p40. However, whether and how miR-10a regulates mucosal DC development and function are largely unknown. In the present study, we found that intestinal lamina propria (LP) DC expressed higher levels of miR10a than that in mesenteric lymph node (MLN) and spleen DC. Retinoic acid (RA), a metabolite of Vitamin A (VitA), has been implicated in regulation of mucosal DC development. Interestingly, expression of ALDH1a, a gene encoding an enzyme converting VitA to RA, was positively associated with miR-10a expression in DC from LPDC, MLN DC and splenic DC. Treatment with RA up-regulated DC expression of miR-10a in vitro. Feeding mice diets with high levels of VitA (VAH) promoted, while feeding VitA deficient diets (VAD) decreased, LPDC expression of miR-10a. By using the luciferase assay, we identified CD11c as a target of miR-10a. In DC specific miR-10a deficient (CD11cCre miR10afl/fl, cKO) mice, CD11c+ DC, especially CD11c+CD11b+ DC, were significantly decreased in intestinal LP. When coculture with microbiota antigen specific T cells, cKO DC promoted the differentiation of T cell to IFNg+ Th1 cells but inhibited Foxp3+ Treg cell development. Collectively, our data demonstrated that miR-10a regulates mucosal DC development by targeting CD11c, and DC expression of miR-10a differentially regulates Th1 and Treg cell differentiation.