bluuebunny/crossref_metadata_2025 huggingface dataset:
Fasciola hepatica Fatty Acid Binding Protein (Fh12) inhibits the activation of murine dendritic cells.
Authors: Ruiz-Jiménez Caleb , Celias Daiana , Silvane Leonardo , Cervi Laura , Espino Ana M.
Published: January 2023

Fasciola hepatica, is a helminth that excretes-secretes different proteins that modulate cells of the immune system, generating anti-inflammatory responses. It has been demonstrated that Fh12 achieves this anti-inflammatory effect by inhibiting the expression of TLR4 induced by LPS in macrophages. Fh12 target the CD14 co-receptor, thus blocking the LPS-CD14 binding, which stop the entire TLR4 activation cascade from the beginning of the LPS-stimuli. Concurrently, Fh12 suppress the phosphorylation of various kinases (p38, JNK and ERK) downstream the TLR4 signaling cascade and induce the activation of macrophages by an alternative pathway. Dendritic cells (DCs) are antigen-presenting cells that play a key role at early phase of innate immunity and that are essential in the development of adaptive immune response. The main objective of the current study was to investigate the effect of Fh12 on the activation of murine DCs matured in the presence of LPS. DCs were isolated from bone marrow of naïve C57BL/6 mice, cultured, differentiated and stimulated in vitro with Fh12 1h prior to stimulation with LPS for 18 hrs. ELISA was used to quantify the amount of a panel of secreted cytokines in culture supernatant. FACS was used to determine the activation of MHCII and co-stimulatory molecules. Results demonstrated that Fh12 significantly inhibits the production of IL-12 and IL-6 and reduced the expression of MHCII and co-stimulatory molecules like CD80 on DCs surface. All assays have a significant P value < 0.05. These results suggest that Fh12 exerts a strong suppressive effect on activation of DC cells, which could have relevant implications in the subsequent development of adaptive immune response to microbial pathogens.
HIV myeloid derived suppressor cells (MDSC) are defective in innate immunity to Mycobacterium bovis BCG
Authors: Garg Ankita , Spector Stephen A
Published: January 2023

Disseminated BCG disease and reduced BCG-specific immunity in HIV infected individuals is often fatal in HIV/TB endemic regions. We previously showed that the immune suppressive CD14+ MDSC expand during in vitro or in vivo HIV-infection. Here we examined the factors responsible for defective anti-BCG immunity associated with HIV MDSC. PBMCs from HIV-uninfected healthy donors were cultured with inactivated HIVBaL. After 5 days CD14+HLA DRhi (DRhi) and HLA DR−/lo (MDSC) cell subsets were sorted by flow cytometry (FACS) and infected with BCG at MOI 5 with or without 25OH or 1,25OH vitamin D (vitD). When infected with BCG, DRhi compared to MDSC produced less IL-10 (p=0.04), and more TNF-α (p=0.02) and IL-1β (p=0.05). FACS analysis showed that MDSC compared to DRhi expressed comparable levels of nVDR (p=0.01), but low levels of CYP27B1 (p=0.003) and cathelicidin (p=0.01). The expression of CD86 on MDSC compared to DRhi in response to BCG was low (p=0.02), and remained low when MDSC were cultured in the presence of 25 OH (p=0.03). The expression of CD86 on BCG infected MDSC was comparable to that of BCG infected DRhi only when cultured in the presence of 1,25OH (p=0.07). Similarly, expression of HLA DR on MDSC compared to DRhi in response to BCG was low (p=0.04) and continued to be low in presence of 25OH (p=0.001). The expression of HLA DR on BCG infected MDSC was comparable to BCG infected DRhi only in the presence of 1,25OH (p=0.43). Finally, intracellular replication of BCG in MDSC isolated from HIV-infected individuals was higher compared to replication in DRhi cells (p=0.02). These findings suggest that the impaired killing of BCG by HIV MDSC is due to their defective vitD-cathelicidin mediated innate anti-mycobacterial activity
The protective role of γδ T cells in neonatal influenza
Authors: Guo Xizhi , Thomas Paul Glyndwr
Published: January 2023

Influenza infection is a continuing worldwide health threat, especially in infants and the elderly. Neonates are highly susceptible to influenza infection, which is often causing hospitalization. γδ T cells are the first T cells to develop during embryogenesis and are a significant component of the neonatal immune system. In the neonatal period, conventional αβ T cell responses are functionally less prominent. Hence, γδ T cells are poised to play a critical role in neonatal infections, however, the specific role of γδ T cells in neonatal influenza infection remains to be elucidated. Using a neonatal mouse model of intranasal influenza infection, we observed the activation and proliferation of γδ T cells within two days after infection. By utilizing Nur77-GFP reporter mice, we demonstrated that the activation of γδ T cells was via the TCR and its downstream signaling. IL-17-producing γδ T cells, rather than IFN-γ-producing cells, dominated in the response. A comparison between wild-type and TCRδ-deficient neonates identified a protective function of γδ T cells in neonates, as reduced weight loss and an increased survival rate were observed in the wild-type compared to knockout animals. In infected lungs, wild-type neonates also had more robust IFN-β production and increased inflammatory cytokine production, especially among the Type 2 cytokines, than the TCRδ-deficient animals. Consistently, more eosinophils infiltrated into the lungs of wild-type neonates. Our findings demonstrate a vital role for γδ T cells in mediating protection during neonatal influenza virus infection via a mechanism which appears to include promotion of a Type 2 immune environment that may provide a new therapeutic target for pediatric influenza treatment.
Central and local adaptations sustain Type I Interferon exhaustion during chronic viral infection
Authors: Jo Yeara , Macal Monica , Dallari Simone , Zuniga Elina
Published: January 2023

During chronic infections, sustained cell adaptation via functional exhaustion has been mostly studied in the adaptive immune compartment but much less is known on how innate immune cells adjust to a persistently infectious milieu. Thus, to understand how innate cells adapt to persistent infections, we studied plasmacytoid dendritic cells (pDCs), which specialize in Type I Interferon (IFN-I) production and often become functionally exhausted in chronic settings. Using a murine chronic viral infection model we demonstrated that pDC IFN-I exhaustion is caused by concerted events at central (bone marrow) and local (spleen) compartments. On one hand, undifferentiated bone marrow pDC progenitors exhibited quantitative and qualitative defects and failed to generate functional pDCs ex vivo. Analysis of transcription and chromatin landscapes via RNA-seq and ATAC-seq revealed that IFN-I signaling pathway was enhanced in bone marrow pDC progenitors. Consistently, IFN-I receptor blockade restored the quantitative (albeit not the qualitative) defects of bone marrow pDC progenitors, which was accompanied by restoration of E2-2, a transcription factor critical for pDC development. On the other hand, splenic pDCs received sustained TLR7 signaling, which promoted their maturation but also rendered them unable to produce IFN-I upon ex vivo TLR re-stimulation or in vivo secondary infection. RNA-seq and ATAC-seq analysis in splenic pDCs revealed significant alterations in multiple pathways and transcriptional regulators that are currently being studied. Our work provides an anatomical, cellular and molecular framework to understand how innate immune exhaustion can be triggered and sustained during a chronic viral infection.
Bradykinin mediates the secretion of coagulation factor VIII by mouse dendritic cells
Authors: Clement Cristina C. , Follenzi Antonia
Published: January 2023

Recent experimental evidences proved that kinins, which are a family of octa- to decapeptides structurally related to bradykinin (BK), have modulatory effects on multiple players of the immune system, including macrophages, dendritic cells (DC), T and B lymphocytes, and induce the activation, proliferation, migration, and effector functions of these cells. To further understand the molecular mechanisms underlying bradykinin mediated activation of DCs, we have employed global proteomics and transcriptomic (mRNA) profiling of mouse DCs activated by BK peptide (RPPGFSPFR). The proteomics profiling was performed using a combination of label-free nanoLC-ESI MS/MS and 2D-DIGE analysis. C57BL/6J wild type mice were treated with: 1) BK, or 2) Captopril, an ACE inhibitor that enhances the endogenous BK, or 3) a combination of both drugs (BK+CAP). Following treatment, the DCs (CD11c+) were purified by immunomagnetic sorting. Comparative Ingenuity Pathway Analysis (IPA) identified the upregulated cellular pathways in the BK/CAP/BK+CAP treated DCs, highlighting molecules involved in the DCs migration/chemotaxis, MHC-I and MHC-II expression, antigen presentation pathways, inflammation and cytokines secretion. Moreover, both the comparative proteomics and the transcriptomic analyses demonstrated the significant upregulation of the coagulation pathway (e.g. coagulation factors V, VII, VIII and PAR2 and PAR3 receptors) in the purified DCs from treated mice. Production of coagulation factors V and VIII by BK-activated DCs was validated by RT-PCR, and by immunofluorescence. Our research highlights novel cellular pathways that regulate the cross-talk between inflammation and coagulation in the bradykinin activated DCs.
Reprogramming of inflammatory monocytes by autophagy modulator LiCl
Authors: Rahtes Allison A , Li Liwu
Published: January 2023

Chronic inflammation is an underlying feature of a variety of highly prevalent diseases such as diabetes, atherosclerosis, inflammatory bowel disease, and cancer. Subclinical endotoxemia has emerged as a common factor correlated with these diseases, potentially contributing to the establishment of chronic inflammation through skewing non-resolving inflammatory monocytes. A better understanding of the underlying mechanisms for the programming of non-resolving inflammatory monocytes may facilitate effective strategies to treat diverse inflammatory diseases. We have observed that subclinical low dose LPS may enable non-resolving inflammatory monocytes through disrupting lysosome fusion and causing accumulation of inflammatory mediators such as p62 and pellino1. Administration of autophagy enhancers such as LiCl can attenuate the effects of low dose LPS, by reducing the levels of pellino1. Together, our data suggest an inhibition of autophagy in response to chronic exposure to subclinical endotoxin. Autophagy is a widely recognized homeostatic cellular process with anti-inflammatory capacity. Further elucidation of the mechanism behind this inhibition may shed light on the link between chronic inflammation and chronic subclinical endotoxin exposure, and provide new avenues for disease treatment.
HECT E3 Ubiquitin ligase Nedd4 is required for anti-fungal innate immune response
Authors: Nuro-Gyina Patrick Kwadwo , Tang Juan , Tang Rong , Zeng Qiuming , Ouyang Song , Guo Hui , Lin Guoxin , Tang Na , Liu Jishi , Zhang Jian
Published: January 2023

Candida albicans is the most common cause of fungal infections in humans, and disseminated candidiasis has become one of the leading causes of hospital-acquired blood stream infections with 45% to 75% mortality rate. The understanding of the host–pathogen interactions and the mechanisms of immune regulation against fungal spread is critical for developing immune-based strategies to combat candidemia. Nedd4 (neuronal precursor cell-expressed developmentally down-regulated 4) is a HECT-type E3 ubiquitin ligase which has been shown to positively regulate T cell activation and proliferation. However, the role of Nedd4 in innate immunity is completely unknown. To elucidate the role of Nedd4 in innate immune response against fungal infection, we generated the mice bearing LoxP-flanked alleles encoding Nedd4 with Rosa26-Cre-ERT2 mice to generate Nedd4fl/flRosa26-Cre-ERT2 mice (called ‘Nedd4CreER mice’ here). We also generated mice deficient for Nedd4 in dendritic cells (DC) (Cd11c Cre.Nedd4f/f) or macrophages (LysM Cre.Nedd4f/f). We found that although Nedd4 might not regulate the signaling via TLRs and Dectin-1, Rosa26-Cre-ERT2 mice treated with tamoxifen or mice deficient for Nedd4 in the myeloid cell lineages (macrophages and dendritic cells) are highly susceptible to systemic C. albicans infection, which correlates with defective pro-inflammatory cytokines in the sera, impaired leukocyte recruitment to the kidneys, defective ROS expression by the granulocytes in the kidneys, and heightened kidney fungal burden. Therefore, our data suggest that Nedd4 expression in the myeloid cells is crucial for C-type lectin receptor (CLR)-mediated innate immune response against systemic C. albicans infection.
Innate immune response in acute lung injury following hemorrhagic shock
Authors: Chu Xiaogang , Subramani Kumar , Warren Marie , Raju Raghavan Pillai
Published: January 2023

Trauma is the major cause of death for Americans under the age of 46 years. Hemorrhagic shock accounts for up to 40% of trauma-related deaths. Hemorrhagic shock evokes an acute, non-specific, systemic inflammatory response syndrome (SIRS) resulting in the damage to multiple organs. Acute lung injury (ALI) is one of the most serious complications in traumatic patients, however, the immunological mechanisms in ALI are still not well understood. Recently studies suggest that mitochondria play an important role in physical injury, leading to the onset of the SIRS. In order to address the mechanistic basis of ALI following hemorrhagic shock, we subjected rats to severe hemorrhage and shock (hemorrhagic injury - HI) or sham procedure and lung tissue was tested. We demonstrate that NLRX1, a mitochondria-targeted protein, that negatively regulates innate immunity and cell death responses, was markedly decreased in HI lung. The decrease of NLRX1 was concomitant with the activation of NF/κB and TBK1 signaling pathways. Lung histochemistry was used to establish pathologic effect on the lung. Significant increases in pulmonary expression of inflammatory signaling molecules IL-6, IL-1β, IL-10, TNF-α and MIP-1α further confirmed HI induced pulmonary inflammation and injury. Our results suggest NLRX1 participate in the pathogenesis of HI related pulmonary diseases and support its important role in the regulation of innate immune response following hemorrhagic shock.
IL-12p35 Inhibits Neuroinflammation and Ameliorates Autoimmune Encephalomyelitis
Authors: Choi Jin Kyeong , Dambuza Ivy M , He Chang , Yu Cheng-Rong , Uche Anita N , Mattapallil Mary J , Caspi Rachel R , Egwuagu Charles E
Published: January 2023

Multiple sclerosis (MS) is an inflammatory demyelinating disease in which cytokines produced by immune cells that infiltrate the brain and spinal cord play a central role. We show here that the IL-12p35 protein might be an effective Biologic for suppressing neuroinflammatory responses and ameliorating the pathology of experimental autoimmune encephalomyelitis (EAE), the mouse model of human MS. We further show that IL-12p35 conferred protection from neuropathy by inhibiting the expansion of pathogenic Th17 and Th1 cells and inhibiting trafficking of inflammatory cells into the brain and spinal cord. In addition, in vitro exposure of encephalitogenic cells to IL-12p35 suppressed their capacity to induce EAE by adoptive transfer. Importantly, IL-12p35 induced the expansion of Treg and Bregs in part through the inhibition of IL-6- and/or IL-27-mediated activation of STAT1 and STAT3 pathways, respectively. Moreover, IL-12p35 inhibited lymphocyte proliferation by suppressing the expressions of cell-cycle regulatory proteins. Taken together, these results suggest that IL-12p35 can be exploited as a novel Biologic for treating CNS autoimmune diseases and offers the promise of ex-vivo production of large amounts of Tregs and Bregs for immunotherapy.
An Integrative Bioinformatics Approach Identifies In Vivo Validated Drug Candidates with Novel Mechanisms of Action in Rheumatoid Arthritis
Authors: Noblin Elizabeth S , Daugherty Aaron C , Farrington Carl , Hakim Isaac , Radin Andrew M , Chua Mei-Sze , Rabe Mark , Fernald Guy , Ford Daniel , Sirota Marina , Schaevitz Laura , Radin Andrew A
Published: January 2023

Rheumatoid arthritis (RA) is an area of active drug development, with over 100 candidates in clinical trials. However, most act on a small number of immunomodulatory targets. Drug candidates that act through new targets or mechanisms could expand treatment options for RA. We applied a data-driven bioinformatics approach and in vivo screen to identify and test new drug candidates and targets that could form the basis of future drug development in RA.

A computational model of RA was constructed by integrating patient gene expression data, molecular interactions, chemical structures, and clinical drug-disease associations. Candidates were scored based on their predicted efficacy in the computational model. FDA-approved treatments for RA were significantly enriched among the top-ranked candidates. Ten high scoring novel candidates were then screened in the collagen-induced arthritis model of RA in rats. Therapeutic treatment with three candidates significantly reduced ankle size, alleviated limb inflammation, improved joint histopathology, and reduced mobility impairments tracked by a novel digital motion endpoint. These candidates are currently approved for metabolic, allergic, and psychiatric indications, and do not act on common RA therapeutic targets. However, links between known candidate pharmacology and pathological processes in RA suggest hypothetical mechanisms that could contribute to efficacy.

Future studies will inform the druggable targets, pathways, and mechanisms that could contribute to each candidate’s efficacy in RA. The candidates could themselves be modified and optimized to increase efficacy in RA. Novel targets identified in these studies could also be the basis of new drug discovery initiatives.
TLR8/7 combined withTLR3 or TLR4 enhances DC-NK driven effector T cells
Authors: Nouri-Shirazi Mahyar A , Tamjidi Saba , Nourishirazi Erika
Published: January 2023

Background

Most current prophylactic vaccines confer protection primarily through humoral immunity. Indeed, aluminum salts that have been used extensively as adjuvants primarily enhance Th2-driven antibody responses. Therefore, new vaccines formulation is moving toward a careful selection of adjuvants that also elicit significant Th1 responses or CTLs. Various TLR ligands have been tested as potential adjuvants in clinical and preclinical studies with some efficacy. These studies suggest that combining more than one of TLR ligands enhance the magnitude of immune responses to cancer and infectious disease. In order to evaluate the synergic effect of TLR agonists for effective induction of cellular immunity, we investigated the effects of individual and/or combined TLR agonists on monocyte-derived DC maturation, DC-NK cross talk and ultimately T cells differentiation.

Results

Among the TLR agonists tested, we found TLR8/7 in combination with TLR3 or TLR4 synergistically optimize DC maturation and IFN-γ secretion from NK cells co-cultured with DCs. Moreover, co-culture of DC-NK-T treated with TLR8/7 and TLR3 or TLR4 increased the frequency of IFN-γ producing effector T cells when compared to Aluminum salt-treated cultures.

Conclusions

Our data suggests that enhancement of a desired cellular immunity requires a selection of TLR agonists with defined synergistic effects. NIH-NIAID 1R03AI103750-01A1
Anti-cotinine CAR-modified T cells provides a novel switchable CAR platform using cotinine-conjugated adaptor molecules
Authors: Park Hyung-Bae , Kim Hyori , Oh Yu Mi , Kim Ki-Hyun , Lee Ji Eun , Chung Junho , Choi Kyungho
Published: January 2023

T cells engineered with chimeric antigen receptor (CAR T cells) have been reported to provide potent anti-tumor effects in various preclinical experimental settings. Subsequently, CAR T cells against some hematologic malignancies showed favorable results in early clinical trials. However, there are various obstacles to overcome in generalizing this CAR T platform. Usually CAR T cells can target only a single tumor antigen although generation of CAR T cells takes considerable labor and expenses. Also, excessive activation of CAR T cells often leads to adverse side effects that need to be controlled. Switchable CAR T platforms using anti-hapten CAR T cells and hapten-conjugated adaptor antibodies was proposed as a novel way to target multiple antigens using single CAR T cells. Also, removal of hapten-conjugated adaptor antibodies can limit adverse side effect caused by CAR T cell recognition of the antigen. Here, we report a novel switchable CAR T platform using a chemical hapten, cotinine. Anti-cotinine CAR T cells generated using anti-cotinine single chain Fv recognized cotinine-conjugated adaptor molecules such as aptamers and antibodies. Especially, anti-cotinine CAR T cells complexed with cotinine-conjugated antibodies could recognize tumor antigens on the surface of tumor cells, which led to activation of CAR T cells and resulting cytotoxicity on tumor cells. Thus, we propose that anti-cotinine CAR T platform may provide useful tools for a novel multi-targeting CAR T therapy.
Role of necroptotic cancer cells in anti-cancer immunity
Authors: Krysko Dmitri , Aaes Tania Løve , Vandenabeele Peter
Published: January 2023

Successful immunogenic apoptosis in experimental cancer therapy depends on the induction of strong host anti-tumor responses. Given that tumors are often resistant to apoptosis, it is important to identify alternative molecular mechanisms that elicit immunogenic cell death.

Necroptosis is one form of regulated necrosis and is mediated by RIPK1, RIPK3, and its substrate mixed lineage kinase domain-like and has been reported to contribute to inflammation under pathological conditions. Necroptosis is commonly induced by ligand-dependent activation of certain members of the TNFR and TLR families. To circumvent the use of cell death-inducing ligands or other immune active molecules that would not allow us to evaluate the immune response resulting uniquely from necroptosis induction, we opted for the use of inducible Tet-On systems for the expression of the downstream necroptotic effector proteins, allowing us to induce necroptosis independently of any receptor activation. In our genetic model a direct dimerization of FADD combined with inducible expression of RIPK3 promotes necroptosis. We report that necroptotic cancer cells release DAMPs and promote maturation of dendritic cells, the cross-priming of cytotoxic T cells, and the production of IFN-γ in response to tumor antigen stimulation. Using both FADD-dependent and FADD-independent RIPK3 induction systems, we demonstrate the efficient vaccination potential of immunogenic necroptotic cells. Our study broadens the current concept of immunogenic cell death and opens doors for the development of new strategies in cancer therapy.
Dissecting the role of Phosphotidylinositol 3-Kinase (PI3K) δ in the germinal center reaction through a mouse model of hyperactivated Pik3cd
Authors: Preite Silvia , Cannons Jennifer L. , Gomez-Rodriguez Julio , Kapnick Senta M. , Handon Robin , Reilley Julie , Notarangelo Luigi , Uzel Gulbu , Schwartzberg Pamela L.
Published: January 2023

The catalytic p110δ subunit of PI3K drives AKT pathways orchestrating activation and differentiation of T and B cells. Patients with gain of function mutations in Pik3cd (encoding for p110δ) exhibit a primary immunodeficiency characterized by lymphopenia, lymphadenopathy, recurrent and chronic infections, and occasionally lymphoma. They have reduced circulating naïve and increased effector T cell numbers, fewer class switched memory B cells and inefficient responses to vaccination. Nonetheless, in patient peripheral blood we find increased frequencies of T follicular helper (Tfh) cells. We have generated a mouse model expressing constitutively active p110δ that recapitulates many features of the human disease. Pik3cdMut mice, both in steady state condition and after immunization, display increased Tfh and germinal center (GC) B cell numbers in secondary lymphoid organs. Moreover, naïve mutant transgenic T cells (OT-II) preferentially acquire Tfh cell phenotype when adoptively transferred to WT mice, indicative of a T cell intrinsic defect. Nonetheless, despite increased Tfh and GC B cells, antigen specific B cell responses are reduced, particularly antigen-specific IgG. In contrast, we find the appearance of autoantibodies in Pik3cdMut mice. We further find that mutant OT-2 cells differentiate to Tfh cells independently from ICOS-ICOSL interactions. In line with this, mutant T cells show higher phosphorylation and inactivation of FoxO1, potent inhibitor of Tfh cell differentiation, that is phosphorylated and inhibited by AKT activation downstream of ICOS. Our results demonstrate that hyperactivated PI3Kδ leads to increased generation of Tfh cells and germinal centers, yet with defective in vivo function.
MicroRNA-21 in Natural Killer T cell α-galactosylceramide activation
Authors: Frias Adolfo B , Buechel Heather Marie , Piccirillo Ann Renee , D’Cruz Louise M
Published: January 2023

Natural Killer T (NKT) cells are innate-like lymphocytes that rapidly produce cytokines upon activation. However, following primary stimulation with the potent ligand α-galactosylceramide (αGC), NKT cells become hyporesponsive, defined as a severe reduction in effector cytokines upon re-stimulation. The purpose of this project is to investigate the role of microRNAs (miRNAs) during NKT cell transition from effector response, expansion and hyporesponsiveness. MiRNAs are ~22bp non-coding RNAs that associate with the RNA-induced silencing complex (RISC) to inhibit the translation of target transcripts. We performed a Nanostring microRNA analysis to determine global expression of microRNAs in NKT cells after α-GC stimulation. MicroRNA-21 (miR-21) was found to be highly upregulated in splenic NKT cells at 3-hours, 3-day, and 30-days post-activation compared to unstimulated controls. Our preliminary data show that MiR-21KO/KO splenic and liver NKT cells have a lower frequency at day-3 compared to WT-littermate controls. We hypothesize that miR-21 regulates the NKT cell transition from activation to hyporesponsiveness and that miR-21KO/KO NKT cells exhibit an interrupted hyporesponsive state.
Effector CD4+ T cells can convert to a CD8+, MHCII-restricted T cell lineage
Authors: Wong Elizabeth , Nyanhete Tinashe , Frisbee Alyse , Ni Qingshan , Zhang Baojun , Payne Tamika , Liu Siqi , Li Chaoran , Chen Liang , Zhuang Yuan , Wan Ying , He You-Wen , Moody Tony , Tomaras Georgia D , Li Qi-Jing
Published: January 2023

CD4+ and CD8+ T cells are conventionally considered as separate lineages characterized by distinct surface markers, function and MHC restriction. However, CD4+ T cells have a recently-appreciated capacity to acquire CD8+ T cell-like characteristics upon TCR-mediated activation. We have identified a novel, CD4+ T cell-derived effector population in mice that is CD4−CD8αβ+ and MHCII-restricted. These cells express a unique lineage-intermediate transcriptional program (ThPOKlo-int Runx3hi-int), upregulate granzyme B/IFN-γ expression and exhibit antigen-specific cytotoxicity. Following acute infection with LCMV Armstrong in wild-type mice or with Listeria monocytogenes in MHCII-restricted, TCR-transgenic mice, we have used CD4+ T cell lineage tracing, MHCII tetramer staining and TCR repertoire sequencing techniques to demonstrate that ex-CD4+ T cell responses can be induced in vivo and originate from bona fide CD4+ T cells. We also report ex-CD4+-like T cells in human HIV patients that are CD8+ and exhibit MHCII-blockable antiviral activity. This population is diminished in elite controller patients upon loss of virus control, indicating that it may contribute to protection against HIV. Finally, we have generated preliminary evidence that ex-CD4+ T cell generation is controlled by an autophagy-related mechanism. CD4+ T cell-specific deficiency in either of the autophagy regulators Vps34 or Atg7 results in dramatic loss of CD4 expression upon antigen stimulation, concomitant with skewing towards the CD8+ transcriptional program in Vps34-deficient cells. We propose that the ex-CD4+ T cell lineage dramatically extends the paradigm of αβ T cell lineage plasticity and may indicate new antiviral vaccine strategies.
Lipopolysaccharide-induced CD300b receptor binding to Toll-like Receptor 4 alters signaling to drive cytokine responses that enhance septic shock.
Authors: Voss Oliver H. , Krzewski Konrad , Coligan John E.
Published: January 2023

LPS, present in gram-negative bacteria membranes, causes strong immune responses following detection by TLR4 on immune cells. Excess immune cell activation resulting from severe infection initiates a pro-inflammatory cytokine storm that leads to a more severe immunopathology, like septic shock, and subsequently, death. Intriguingly, Cd300b−/− mice were observed to be more resistant to septic shock than WT mice, however the mechanism responsible for this phenomenon remained elusive. Here, we identify LPS as a ligand for CD300b, and demonstrate that CD300b-expressing macrophages (Mφ) are key cells augmenting septic shock.

We show that CD300b and DAP12, associate with TLR4/CD14 upon LPS binding, resulting in the dissociation of MyD88/TIRAP from the complex and the recruitment and activation of Syk and PI3K. This leads to a PI3K/AKT-dependent inhibition of the MEK1/2-ERK1/2-NFκB pathway, subsequently resulting in a reduced IL-10 production by Mφ. In addition, CD300b also enhances TLR4/CD14-TRIF-IRF3 signaling, resulting in elevated IFN-β levels. Compared to WT mice, Cd300b−/− mice demonstrate decreased serum levels of pro-inflammatory cytokines and elevated levels of the IL-10 anti-inflammatory cytokine. Neutralization of IL-10 in Cd300b−/− mice diminishes their survival advantage over WT animals. Importantly, we demonstrate that an anti-CD300b Ab treatment suppresses pro-inflammatory cytokine and enhances anti-IL-10 production in Mφ, thus reducing the mortality of septic mice.

These findings provide evidence for a previously unappreciated regulation of TLR4 signaling by CD300b in myeloid cells, and highlight CD300b as a potential therapeutic target for clinical intervention to manage septic shock in humans.
Influence of aging on the phenotype and function of alveolar macrophages
Authors: Lafuse William P , Rajaram Murugesan VS , Curry Heather M , Nealer Connor , Canan Cynthia H , Moliva Juan I , Torrelles Jordi B , Turner Joanne , Schlesinger Larry S
Published: January 2023

Aging is associated with a decline in immune function (immunosenescence), resulting in increased susceptibility to infectious diseases, malignancies, and autoimmunity. However, the immunosenescence paradigm does not explain increased circulating pro-inflammatory cytokines and increased tissue expression of pro-inflammatory genes with aging. These observations have led to a second paradigm, termed inflammaging, in which chronic, low grade inflammation develops with increasing age. Published studies have shown that inflammaging also occurs in the lung. However, the influence of inflammaging on alveolar macrophage (AM) phenotype and function is largely unexplored. In this study, we examined the phenotype and function of AMs isolated from young (3 months) and old (18 months) C57BL/6 mice. Phenotypic analysis of AMs by flow cytometry showed that in young mice there is a major AM population that is CD11c+CD11b− and a minor population that is CD11c+CD11b+. In young mice, this minor population represents 1.3% of AMs. However, in old mice the percentage of this minor population is significantly higher (4.9%, p <0.0001). The CD11c+CD11b+ AMs have higher expression of several cell surface markers, e.g. MHC II and CD64, indicating that these AMs are in a more prime or activated state. Functional studies of these AMs stimulated with LPS, PamCSK4, or infected with Mycobacterium tuberculosis show significant spontaneous production of IFN-β, CCL2, and IL-10 that is prolonged in old mice and increased further by stimulation with ligands or infection. Our studies indicate that inflammaging in the lung primes AMs, resulting in robust production of key cytokines upon stimulation that potentially alter host susceptibility.
Feeding immunity: The regulation of circadian immune responses by mealtimes
Authors: Geiger Sarah Sofia , Traba Javier , Sack Michael N. , O’Neill Luke A. J. , Siegel Richard M.
Published: January 2023

Both, mice and humans have a striking circadian variability in the responsiveness to innate immune stimuli and in mice, susceptibility to sepsis induced by acute stimulation of the immune system with LPS; and disruption of the circadian rhythm is associated with inflammatory diseases, such as cancer and obesity. The intrinsic cellular clock exists in immune cells and can control some aspects of innate immunity, but what extrinsic signals, termed ‘zeitgebers’ or time-givers, entrain the circadian behavior of immune response are not known. We have investigated the relative contributions of light and the feeding cycle to innate immune responses by dissociating the light cycle and access to food through time restricted feeding (TRF), allowing access to food either during the dark - and natural feeding time of mice - or the light phase. Using RNA profiling we found that the circadian clock in the liver is entirely programmed by food, whereas in the spleen, mainly light influences the expression of clock genes. Strikingly, in this setting susceptibility to LPS-induced morbidity was entrained by the food, not the light cycle, suggesting that food is the ‘zeitgeber’ for circadian susceptibility to LPS-induced sepsis. While the exact mechanism is under thorough investigation, daily mortality patterns following LPS stimulation could not be attributed to corticosteroids or ketone bodies, both of which possess anti-inflammatory properties and exhibit strong food dependency. Understanding the interface between nutrition and circadian rhythmicity may provide new insights with which to design strategies to prevent and improve metabolic and inflammatory diseases in individuals at risk.
Palmitic acid promotes t-bet and gata-3 mRNA degradation in NKT cells via regulated IRE1a-dependent decay
Authors: ko jaesung , Koh Jae Moon , So Jae-Seon , Chung Doo Hyun
Published: January 2023

Long chain fatty acids (LCFAs) exert pro-inflammatory effects in vivo. However, little is known regarding the effect of LCFAs on invariant (i) NKT cell functions. Here, we report an inhibitory effect of saturated LCFAs on transcription factors in iNKT cell. Among the saturated LCFAs, palmitic acid (PA) specifically inhibited IL-4 and IFN-g production and reduced gata-3 and t-bet transcript levels in iNKT cells during TCR-mediated activation. In iNKT cells, PA was localized and induced dilation in the endoplasmic reticulum and increased the mRNA levels of downstream molecules of IRE1 RNase. Moreover, PA increased the degradation rates of gata-3 and t-bet mRNA, which were restored by IRE1 inhibition, indicating that gata-3 and t-bet are cleaved via regulated IRE1a-dependent decay (RIDD). A PA-rich diet suppressed IL-4 and IFN-g production by iNKT cells in wildtype mice, thereby attenuating arthritis. This study demonstrates that a saturated LCFA induced RIDD-mediated t-bet and gata-3 mRNA degradation in iNKT cells, thereby suppressing cytokine production.
Investigating modulation of inflammation in atherosclerosis by complement protein C1q
Authors: Pardo Paulina Valeria , Flores Abraham , Wong Ashley J. , Ho Minh-Minh , Fraser Deborah A.
Published: January 2023

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of fatty streaks within arterial walls. In hyperlipidemic conditions, cholesterol accumulates in macrophages disrupting cholesterol trafficking and leading to an inflammatory environment. C1q plays a dual role in atherosclerosis. Activation of complement by C1q exacerbates disease by release of pro-inflammatory activation fragments C3a and C5a. However, we have shown in vitro that C1q has numerous protective roles in atherosclerosis such as improving macrophage cholesterol ingestion and efflux, and polarizing macrophages towards a resolving, anti-inflammatory phenotype. Here we investigate if C1q programs similar macrophage inflammatory responses in vivo. We hypothesize that C1q polarizes macrophages to a protective phenotype in the early stages of atherosclerosis by increasing anti-inflammatory cytokines and decreasing pro-inflammatory cytokines, leading to a reduction in disease markers such as cholesterol and triglyceride levels and atherosclerotic lesion size. To investigate, atherosclerosis model, LDLr−/−, mice that are C1q sufficient or deficient were fed a high fat Western diet for 12 weeks. Changes in plasma cyto/chemokine levels are measured by luminex multiplex assay. Cholesterol and triglyceride levels are measured by colorimetric assay. Atherosclerotic lesions are measured in H&E stained aortic sinus. Preliminary data suggests that C1q modulates disease markers as predicted. By understanding the role of C1q in atherosclerosis it may be possible to design improved treatment strategies.
A novel role for C5a in regulating B1 cell homeostasis and function
Authors: Karsten Christian M , Bröker Katharina , Figge Julia , Manz Rudolf A , Köhl Jörg
Published: January 2023

B1 cells are innate-like B cells producing natural IgM (nIgM) antibodies without previous antigen exposure. They ensure first line humoral defense and long-lasting immunity against T-cell independent antigens found in encapsulated bacteria. B-1 cells are predominantly located inside body cavities and in lower numbers in spleen and bone marrow. Previous findings suggest that their egress is contolled through several integrins and the chemokines CXCL12 and CXCL13. B1 cell need to migrate from the peritoneal cavity to the spleen to become nIgM producing plasma cells. The role of complement in B1 cell homeostasis and migration has not been explored. Here, we determined the impact of C5a and the C5a receptors (C5aR) 1 and 2 on B1 cell biology. We found significantly reduced B1 cell numbers in the peritoneal cavity of C5aR1−/− or C5aR2−/− as compared with wildtype mice that were associated with increased B1 cell numbers in the spleen. Consequently, C5aR1−/− mice had elevated serum levels of nIgM against phosphorylcholine and different polysaccharides of S. pneumoniae under steady state conditions. Using a GFP-C5aR1 reporter mouse, we further demonstrate that C5aR1 expression on B1 cells is upregulated whereas the CXCL13 receptor CXCR5, CXCR4, IgM and aM (CD11b) integrin expression is downregulated before B1 cells leave the peritoneal cavity upon TLR2 stimulation in vivo. In line with this observation, we found that in C5aR1−/− mice, TLR2-driven B1 cell egress was diminished, as B1 cell numbers in C5aR1−/− mice were unchanged whereas they markedly dropped in wildtype mice. Thus, our data suggest a novel role for C5a in the regulation of B1 homeostasis in the peritoneal cavity, nIgM production and TLR-driven egress from the peritoneum into the spleen.
Obesity-induced changes to the immune landscape in human renal cell carcinoma subjects
Authors: Norian Lyse A , Norris Katlyn E , Wald Gal , Thomas Lewis J , Bertrand Laura A , Buchta Claire M , Boi Shannon K , Nepple Kenneth G , Brown James A
Published: January 2023

Immunotherapy for advanced renal cell carcinoma (RCC) has shown tremendous promise. However, clinical responses remain <30% and the reasons are unclear. Obesity affects ~35% of U.S. adults and is a major risk factor for RCC. We reported previously that obesity impairs immunotherapeutic efficacy in a murine renal cancer model, due to increased myeloid suppressor cells and weakened effector CD8 T cell responses. Based on these results, we hypothesized that obesity would also induce detrimental shifts in the immune landscape in treatment-naive subjects with confirmed RCC. In an IRB-approved study, we consented 86 RCC subjects with obesity (mean BMI = 38.4) and 77 normal to over-weight RCC subjects (mean BMI = 25.0), plus tumor-free controls, into a prospective study to examine intra-tumoral and systemic leukocytes by multi-parameter flow cytometry, plasma proteins via multiplex, and tumor gene expression by Nanostring. Numerous systemic cell populations (ex: CD45RO+ PD1+ CD4 or CD8 T cells, and HLA-DR- CD14− CD11b+MDSC, all p>0.05) were unaltered by obesity in RCC subjects. Peripheral CD14+ inflammatory monocytes and Foxp3+ Tregs were decreased in RCC subjects with obesity (both p< 0.05). However, obesity altered plasma proteins to favor RCC progression, with increased angiopoietin, VEGF-A and –C, and IL-8 (all p< 0.05). Within renal tumors, obesity did not alter CD4 or CD8 T cell percentages or phenotypes (CD45RO, PD1, CD56). Nanostring analysis of renal tumors revealed multiple obesity-related changes in pro-tumorigenic genes. Thus, obesity in RCC subjects had surprisingly few effects on cellular immunity, but multiple effects on protein mediators that may shift the overall immune landscape toward one that favors tumor progression.
Understanding the effects of a calorie restriction mimetic on renal cancer progression and CD8 T cell immunity
Authors: Orlandella Rachael M , Boi Shannon , Smith Daniel , Norian Lyse A
Published: January 2023

Metastatic renal cell carcinoma (RCC) exhibits high mortality rates and chemotherapeutic resistance. Immune-based therapies, including high-dose IL-2 and anti-PD1, are efficacious in ~20% of patients; however, both are associated with substantial toxicity. This highlights the need for additional research aimed at improving response rates while also limiting toxicity. Previous findings by other groups have demonstrated that nutrient deprivation prior to chemotherapy reduces treatment toxicity and may improve therapeutic efficacy in cancer patients. Recent murine research shows that diets containing calorie restriction mimetics (CRMs) can recapitulate these beneficial effects by enhancing anti-cancer immunity. Here, we examined the impact of the alpha-glucosidase inhibitor acarbose, a potential CRM, on renal tumor burden and immune profiles. BALB/c mice were fed an acarbose-containing diet or macronutrient-matched control diet for 4 weeks prior to challenge with syngeneic Renca renal carcinoma cells. Although no significant differences were observed in primary renal tumor outgrowth or spontaneous lung metastases (both, p<0.05), acarbose-fed mice exhibited severe weight loss following intra-renal tumor challenge (p=0.02). Tumor-bearing mice fed an acarbose diet displayed increased percentages of total splenic (p=0.03) and intra-tumoral (p=0.04) CD8 T cells versus controls. Future experiments will evaluate the effects of the acarbose CRM on immunotherapeutic efficacy and ensuing immune responses. Our findings demonstrate that further research is required to understand how specific CRMs impact tumor outgrowth and anti-cancer immunity.
Obesity promotes cancer growth by enhancing the suppressive activity of intratumoral myeloid cells
Authors: Al-Ramadi Basel K , Kaimala Suneesh , Mohamed Yassir A. , Al-Sbiei Ashraf , Bashir Ghada , Fernandez-Cabezudo Maria J
Published: January 2023

Epidemiological evidence suggests that obesity is linked to increased risk of developing several types of cancer. However, the mechanisms underlying this phenomenon remain unknown. Successful growth of cancer cells depends on developing mechanisms to evade host immunosurveillance, particularly within the tumor microenvironment. In the current study, we utilized a high-fat-diet (HFD)-induced obese mouse model to gain mechanistic insight into the effect of obesity on progression of implantable syngeneic tumors. When lean or obese mice were implanted with B16.F1 melanoma or MC38 colon carcinoma, tumor growth was more robust in obese group than lean controls, suggesting that obese conditions promote tumor growth. Multi-color flow cytometric analysis and gene expression profiling were carried out on distinct subpopulations of intratumoral myeloid cells. Tumors of lean mice had higher percentages of myeloid cells compared to obese counterparts with characteristics typical of activated inflammatory macrophages. In contrast, tumors of obese mice had disproportionately higher frequency of myeloid derived suppressor cells (MDSC) with a gene signature suggestive of immunosuppressive potential. These findings highlight the role of metaflammation in tumor growth promotion via the regulation of frequency and activation status of distinct subpopulations of intratumoral myeloid cells.
ERK Signaling-regulated Immune Responses that contributes to Malaria Parasitemia and Pathogenesis
Authors: Wu Xianzhu , Dayanand Kiran K. , Thylur Ramesh P , Norbury Christopher C , Gowda Channe
Published: January 2023

Malaria morbidity and mortality represent a major health and social burdens in many countries around the globe. Given that malaria parasites are becoming more and more refractory to several commonly used drugs, a vaccine is needed to control malaria. However, development of an effective vaccine has been hampered due to incomplete understanding of immunological processes and their regulation. We studied the role of ERK1/2 MAPK signaling regulated immune responses by targeting the signaling pathway in P. berghei ANKA-infected mice, an experimental cerebral malaria model. The signaling was targeted using two small molecule inhibitors PD98059 and an FDA approved anti-cancer drug trametinib, both of which specifically inhibit activities of MEK1/MEK2, the upstream kinases of ERK1/2. The results showed that ERK signaling significantly curtails B1 cell expansion, IgM production, phagocytic receptor expression, and phagocytic clearance of parasites, thereby contributing to high parasite burden. The results also showed that ERK1/2 signaling upregulates pro-inflammatory responses and downregulates anti-inflammatory responses, promoting Th1 response and impeding Th2 response. This resulted in increased T cell cytotoxicity and enhanced infiltration of immune cells to the brain, brain vascular damage and dysfunction. Thus, we identified several previously unrecognized immune regulatory processes through which ERK1/2 signaling exacerbates malaria parasitemia and pathogenesis. Our findings highlight how the ERK1/2 signaling-regulated immune responses limit parasite clearance and contribute to severe malaria pathogenesis.
B cells eliminate virus-specific CD8 T cells through a FasL:Fas dependent mechanism during high-dose influenza virus infections.
Authors: Zacharias Zeb Raplh , Legge Kevin L
Published: January 2023

Our prior studies have shown that after high-dose inoculums or infection with high-pathogenic strains of influenza A virus (IAV), the IAV-specific CD8 T cell response is targeted and killed by plasmacytoid dendritic cells (pDCs) within the draining lymph nodes (LN) in a FasL:Fas (pDC:T cells) dependent manner. This limits host immunity and drives severe disease. Although donor pDCs are sufficient to drive this response in FasL defective gld mice, our recent findings show that FasL+ B cells out number FasL+ pDCs 50:1 within the LN during high-dose IAV infections. This suggested that B cells might contribute to the overall loss of CD8 T cells during high-dose/high-pathogenic IAV infections. Further, we have identified at least two subsets of FasL+ B cells (i.e. CD11c+ and CD11c−) that accumulate at higher numbers within the LN following high- vs. low-dose IAV infections. Additionally, our results suggest that FasL expression by CD11c+ and CD11c− B cells is induced in part by IL-12p40, which is expressed at higher levels in the LN during high-dose IAV infections. Finally, adoptive transfer studies utilizing gld host mice demonstrate that both donor CD11c+ and CD11c− B cell subsets can eliminate IAV-specific CD8 T cells in a FasL:Fas dependent manner during high-dose IAV infections. Therefore overall it appears that FasL+ B cells contribute to the loss of IAV-specific CD8 T cells during high-dose IAV infections ultimately leading to the death of the host.