bluuebunny/crossref_metadata_2025_split huggingface dataset:
Constitutive and inducible expression of the neurokinin-1 receptor for the inflammatory neuropeptide substance P in human microglia and astrocytes
Authors: Burmeister Amanda Rae , Johnson Morgan Brittany , Ramesh Geeta , Martinez Alejandra , Philipp Mario , Marriott Ian 
Published: January 2023

Abstract
We have previously demonstrated that murine microglia and astrocytes express the neurokinin-1 high affinity receptor (NK-1R) for the neuropeptide substance P. Importantly, we have also shown that substance P/NK-1R interactions augment the inflammatory responses of isolated murine glia to bacterial challenge and exacerbate in vivo neuroinflammatory damage within the central nervous system in mouse models of meningitis. Here, we show the constitutive expression of NK-1R in acutely isolated non-human primate (NHP) brain tissue and ex vivo brain slices. Interestingly, NK-1R protein levels were increased in brain tissue as rapidly as 4 hours following ex vivo infection with Borrelia burgdorferi, the causative agent of Lyme neuroborreliosis. Furthermore, we observed higher NK-1R protein levels in NHP brain tissue at 2 and 4 weeks following in vivo infection, increases that correlated with decreases in the expression of glial fibrillary acidic protein. These in vivo changes were prevented by treatment with a specific NK-1R antagonist suggesting a key role for substance P/NK-1R interactions in these effects. We then investigated the expression of NK-1R in human glial cells. While human microglia showed robust NK-1R expression at rest, astrocytes constitutively expressed only low levels. However, challenge with bacteria or their products elicited rapid increases in whole cell and cell surface NK-1R protein expression by primary human astrocytes and cell lines. Together, these data indicate that human microglia and astrocytes show constitutive and/or inducible expression of the substance P receptor and may therefore be susceptible to this pro-inflammatory neuropeptide.
Impact of Klebsiella pneumoniae on efferocytosis of polymorphonuclear cells
Authors: Jondle Christopher Ned , Mishra Bibhuti , Sharma Jyotika 
Published: January 2023

Abstract
Efficient clearance of invading pathogen and the infiltrating cells by phagocytic cells are two key events that are required for successful elimination of a pathological insult. Clearance of the pathogen is usually achieved by phagocytosis and dead cells are cleared by a process called efferocytosis. While there is substantial information on various strategies employed by a number of pathogens to subvert phagocytosis, our knowledge about inhibition of efferocytosis by microbes is extremely limited. In that regard, we observed that, polymorphonuclear cells (PMNs) infected with Klebsiella pneumoniae (KPn), an opportunistic pathogen and a frequent cause of nosocomial infections leading to sepsis, were efferocytosed less than their uninfected counterparts. This inhibition did not correlate with the infection induced effect on phosphatidylserine (PS) exposure, a well-studied “eat me” signal recognized by efferocytic cells or p53 expression, as a marker of apoptosis in PMNs. We further observed that bacterial replication is required for the full inhibitory effect on efferocytosis since heat-killed KPn were not as effective in inhibiting efferocytosis of PMNs. Next we examined the possible role of LPS in diminishing effecrocytosis, given that KPn is a Gram-negative bacterium. By utilizing cells deficient in TLR-4 (a receptor for LPS) and direct stimulation with LPS we determined that LPS is able to directly inhibit efferocytosis of PMNs. To our knowledge, this study is the first to show a modulatory effect of KPn infection and LPS on PMNs leading to inhibition of their efferocytic uptake by macrophages. This could have implications in acute and chronic airway inflammatory diseases such as pneumonia, sepsis, cystic fibrosis, and COPD.
Social stress-induced priming of innate immunity is associated with translocation of commensal lactobacilli to the spleen
Authors: Lafuse William P , Gearinger Rachel , Fisher Sydney E. , Bailey Michael T. 
Published: January 2023

Abstract
Exposure to the social disruption (SDR) stressor, which involves repeated social defeat in subordinate mice, increases serum cytokine levels, results in accumulation of CD11b+ myeloid cells in the spleen, and primes macrophages for increased cytokine and microbicidal activity. Our previous studies showed that the intestinal microbiota are necessary for SDR-enhancement of splenic macrophage microbicidal activity. The current study determined whether SDR-induced translocation of microbes to the spleen was associated with changes in spleen myeloid cell populations and cytokine mRNA. The SDR stressor increased the numbers of spleen neutrophils and inflammatory monocytes compared to control mice, while having no effect on numbers of non-classical monocytes and red pulp macrophages. Using an RNA flow cytometry assay, we found that neutrophils and inflammatory monocytes from SDR mice express higher levels of IL-1β mRNA compared to control mice. To detect translocated bacteria, qRT-PCR was used with bacterial group specific primers to 16S rRNA in addition to traditional bacterial cultures. SDR induced the translocation of Lactobacillus to the spleen, but translocation of other taxa, such as Bacteroides and Bifidobacterium, was not increased in stressor-exposed mice. Levels of Lactobacillus 16s rRNA in the spleen positively correlated with increased levels of IL-1β and IL-23 mRNA in stressor-exposed mice. Our results indicate that stressor exposure increases translocation of Lactobacillus from the gut to the spleen and primes the innate immune system for enhanced reactivity. These studies suggest that commensal microbes are key players in stressor-induced immunopotentiation. Funded by NIH grant R21AI107238.
Antigen Presenting Cells as Drivers of Specialized Immune Responses
Authors: Devarajan Priyadharshini , Vong Allen M , Bautista Bianca , Castonguay Catherine H , Swain Susan L 
Published: January 2023

Abstract
We have defined the “Memory Checkpoint” as the period when CD4 effector T cells (TEFF) require cognate antigen (Ag) recognition for further differentiation into CD4 memory T cells. Using an influenza A virus (IAV) infection model, we find that generation of CD4 cytotoxic T cells (ThCTL) from TEFF requires MHC Class-II restricted Ag at this checkpoint, 5–7 days after initial infection. Ag recognition at this checkpoint also determines CD4 follicular helper T cell (TFH) generation in our model. We have used transgenic mouse models in which Ag presentation is restricted to specific antigen presenting cells (APC), bone marrow chimeras and addition of various APC subsets at the memory checkpoint to study which APC drive TEFF to TFH vs. ThCTL. Our studies indicate that distinct APC, during this checkpoint, promote different fates resulting in generation of distinct effector subsets. While, as expected, B cell APC are necessary for TFH responses, the APC subset(s) that promote ThCTL generation are not yet clear. Our data indicate that a specialized hematopoietic subset of CD11c+ APC, that are preferentially localized in the infected tissue, is required at this checkpoint, to support ThCTL generation. Once we identify the APC subset(s) needed, we plan to target Ag to particular APC at the checkpoint, to uniquely promote distinct functional subsets and evaluate their function. We suggest this may allow us to tailor the quality of the effector response to better protect against distinct pathogens.
Plasticity along with differentiation of Tc17 cells is controlled by CTLA-4 (CD152)
Authors: Brunner-Weinzierl Monika C , Lingel Holger , Pick Jonas , Huber Magdalena , Pierau Mandy , Arra Aditya 
Published: January 2023

Abstract
Tc17 cells are known to express low IFN-γ and granzyme B, resulting in diminished cytotoxicity. Adoptive transfer into Ag-bearing hosts converts them partially towards IFN-γ producing phenotype retaining Tc17 characteristics like increased persistence of survival. Nevertheless molecular mechanisms involved in Tc17 lineage plasticity and stability are yet to be identified. CTLA-4, a homologue to co-stimulatory molecule CD28, diminishes cytotoxicity of Tc1 cells by selective downregulation of GranzymeB, Eomes, and IFN-γ production, but its impact on Tc17 differentiation and plasticity is not known. CTLA-4+/+ and −/− CD8+ T cells were stimulated under Tc17 conditions. Tc1 and Tc17 characteristics in these cells were analysed by flow cytometry, western blot and real-time-PCR. For plasticity studies, cells were re-stimulated and analyzed for expression of Tc17 and Tc1 characteristics. Listeria Monocytogenes(LM) infection model and B16 melanoma model were used to investigate the effects of CTLA-4 on Tc17 cell mediated clearance of infection and controlling tumor growth. CTLA-4 showcased a cell intrinsic ability to enhance Tc17 differentiation program, by sustaining the levels of IL-17 inducing factors like STAT3, RORγ T and IRF4. Adoptively transferred Tc17 cells lacking CTLA-4 signal were more effective in clearing LM infection and controlling tumor growth. Re-stimulated CTLA-4 incompetent Tc17 cells revealed that higher cytotoxicity of these cells is due to their enhanced plasticity nature to develop Tc1 characteristics. Inactivation of CTLA-4 could be a unique target for Tc17 cells, to enhance lineage plasticity and develop Tc1 characteristics with increased persistence of survival and immunity.
The fate of CD8+ T cells during infection is linked to their developmental origin
Authors: Smith Norah L , Wang Jocelyn , Nzingha Kito , Reynaldi Arnold , Davenport Miles P , Rudd Brian D 
Published: January 2023

Abstract
Key features of the CD8+ T cell response to infection are the robust clonal expansion and phenotypic diversification of naïve precursors into effector and memory cells. However, mechanisms that regulate the fate of naïve CD8+ T cells after microbial challenge remain a subject of controversy. Previous studies have suggested heterogeneity in the effector pool is driven by differences in TCR avidity, asymmetric cell division, and environmental cues. However, one variable that has not been closely examined is the developmental origin of naïve CD8+ T cells. Notably, the pool of naïve CD8+ T cells in the adult is comprised of clonotypes that were produced during various stages of life. To determine whether the developmental origin of naïve CD8+ T cells in adults plays a deterministic role in their fate after infection, we developed a system to ‘timestamp’ CD8+ T cells from various stages of development (1 day, 1 week, and 4 weeks) and examined their phenotype and behavior in 8 week-old adult mice. Interestingly, we found naïve CD8+ T cells made early in life predominantly display a CD44hi CD122hi memory phenotype whereas cells made later in life are CD44lo CD122lo. In vitro stimulation of stamped populations showed that fetal- and neonatal-derived CD8+ T cells are hypersensitive to IL12/IL18 and proliferate more rapidly than cells made in older animals. In response to infection in adulthood, both polyclonal and monoclonal CD8+ T cells produced in early life skew towards a terminally differentiated short-lived effector phenotype (KLRG1hiCD127lo), indicating this phenomenon is TCR independent. These data indicate that the spectrum of CD8+ T cell differentiation observed after infection is influenced by when the responding cells were initially made.
Slamf6 acts as a negative regulator of NKT cell expansion in the presence of a strong agonist
Authors: DeVault Victoria L , Dienz Oliver , Lilley Graham WJ , Benoit Patrick J , Schwartzberg Pamela L , Boyson Jonathan E 
Published: January 2023

Abstract
Signaling lymphocyte activation marker family member 6 (Slamf6) is a cell surface signaling receptor that plays an important role in NKT cell development. Upon activation with the CD1d ligand α-galactosylceramide (αGalCer), NKT cell Slamf6 expression increases dramatically (~10-fold) and it maintains this high level of expression for at least 5 days after activation. The mechanisms through which Slamf6 regulates NKT cell function are largely unknown. To investigate the effect of Slamf6 on peripheral NKT cell populations, we challenged C57BL/6 (B6) or B6.Slamf6−/− mice with the NKT-specific agonist αGalCer. Examination of liver NKT cell numbers 3 days after challenge revealed a 50-fold increase in B6 mice over vehicle-treated controls. In contrast, we observed a 270-fold increase in NKT cells in B6.Slamf6−/− mice versus controls. An analysis of spleen NKT cells yielded similar results. A comparison of in vivo BrdU uptake by NKT cells between B6 and B6.Slamf6−/− mice revealed no significant differences in proliferation. In addition, NKT cell intracellular IFN-γ, IL-4, and TNF production between B6 and B6.Slamf6−/− mice after administration of αGalCer revealed no difference in the production of these cytokines. We next evaluated the Slamf6-specific effect on NKT cell expansion using an in vitro co-culture system. Cross-linking of Slamf6 on purified B6 NKT cells resulted in diminished NKT cell expansion and increased numbers of TUNEL+ NKT cells. Taken together, these data support a model where Slamf6 acts as a negative regulator of NKT cell expansion in the presence of a strong agonist by regulating the death threshold of NKT cells. These data suggest that Slamf6 blockade could be a useful tool to manipulate the expansion of NKT cells in vivo.
CD4+ T Cell Differentiation is Determined During an Initial Priming Phase and Occurs Independently of Effects due to Division or Survival.
Authors: van Panhuys Nicholas J , Palmer Douglas 
Published: January 2023

Abstract
The differentiation of naïve CD4+ T cells into discrete effector subsets, each able to mediate specific aspects of immunity is a central tenet of the adaptive immune response. Current dogma suggests that the generation of committed effector CD4+ T cells takes place over a 3–4 day period following initial engagement of the TCR. Activation of CD4+ T cells is thought to occur in three distinct functional phases, priming, proliferation and differentiation. Here, we present data indicating that CD4+ T cells acquire the disposition to commit to either Th1, Th2 or Treg lineages very soon after activation, prior to the induction of division. Further, we show that division is not required for the induction of a full program of differentiation to occur. We demonstrate that TCR signaling is responsible for two distinct activation programs whereby the strength of signal that a naïve T cell receives works in concert with cytokines at an early phase to induce differentiation, whereas the length of TCR signal can be construed as a secondary component of activation which controls the ability of CD4+ T cells to enter into division.
Absence of preferential high-affinity TCR expansion during CD4 T cell responses
Authors: Martinez Ryan J , Andargachew Rakieb , Martinez Hunter , Evavold Brian D 
Published: January 2023

Abstract
Current views assume higher-affinity T cells, as identified by pMHCII tetramers, possess enhanced capabilities while lower-affinity T cells are inconsequential to the immune response. We set out to formally address the contribution of low-affinity T cells to the expanded repertoire. To perform this study we employed a micropipette based assay and T cell receptor signaling functional reporter to quantify the total number of antigen-specific CD4 T cells from naïve precursors through their expansion to antigen. In the naïve state, an in vivo limiting dilution assay revealed hundreds more precursor T cells than defined by pMHCII tetramers, finding high-affinity T cells comprise only 5–30% of the total naïve repertoire. Upon immunization, low-affinity T cells expand similarly to higher-affinity counterparts, demonstrating no affinity maturation or preferential expansion of the highest-affinity T cells as the immune response progressed. These findings demonstrate affinity diversity of CD4 T cells is maintained without a selective shift favoring the highest-affinity cells.
Reciprocal regulation of IL-17 and IL-22 secretion by CD4 T cells through the Aryl Hydrocarbon Receptor
Authors: McAleer Jeremy , Lewis Alisha Milton , Nguyen John 
Published: January 2023

Abstract
CD4 T cells are critical for host defense against microorganisms and direct immune responses by differentiating into specialized subsets. While Th17 cells secreting IL-17 and IL-22 contribute to vaccine immunity, they have also been implicated in autoimmune inflammation. Our goal is to understand how endogenous factors regulate Th17 cell cytokine secretion. Murine models have demonstrated that the aryl hydrocarbon receptor (AhR) can directly promote Th17 cell differentiation or provide systemic anti-inflammatory effects, suggesting that pleiotropic functions of AhR may be dependent the ligand and/or cell type studied. Tryptophan metabolites have been identified as endogenous AhR agonists that can modulate T cell immunity. Here, we examined a potential link between tryptophan metabolism and the AhR on cytokine secretion by primary human CD4 T cells cultured under Th17 conditions. L-tryptophan decreased IL-17 secretion and RORgt expression in T cells, but did not significantly affect IL-22 secretion. Similar results were observed with the tryptophan metabolite L-kynurenine. Conversely, AhR blockade decreased IL-22 with no effect on IL-17 secretion. These data suggest that IL-17 and IL-22 production are reciprocally regulated in human CD4 T cells through tryptophan availability and the AhR, and provide an avenue for therapeutic modulation of pro-inflammatory T cell responses.
‘Efferocytosis of infected apoptotic cells inhibits Th17 differentiation via PGE2-EP4 signaling’
Authors: Dejani Naiara , Fortino Felipe , Nino Victoria , Medeiros Alexandra Ivo 
Published: January 2023

Abstract
Clearance of infected apoptotic cells (IAC) by dendritic cells (DC) triggers the production of Th17-related cytokines. Moreover, we have found that prostaglandin E2 (PGE2) is also produced during efferocytosis containing microbial products, but the effect of this prostanoid on Th17-differentiation is still unknown. The purpose of this study was to evaluate the role of PGE2, produced during recognition of IAC by DC, on Th17 differentiation. As source of IAC, Raw 264-7 cells were cultured with Escherichia coli during 2h for phagocytosis and apoptosis was induced by UV irradiation. BMDC were co-culture with IAC during 18h, and the supernatant from this co-culture was used to differentiate naïve CD4+ T cells for 4d, plus anti-CD3/anti-CD28. Phagocytosis of IAC by DC promotes the production of high levels of PGE2 (15±0.7 ng/ml), IL-6 (26±0.4 ng/ml), TGF-β (3.5±0.09 ng/ml) and IL-1β (5.4±0.1 ng/ml), when compared to phagocytosis of uninfected-AC. The treatment with COX inhibitor significantly decreased PGE2 level and promotes Th17 cell differentiation at least 4 times higher (16%) than untreated co-cultures (4%). Moreover, adding back exogenous PGE2 decreased drastically the percentage of Th17 cells (2%). Moreover, the treatment of naive T cells with antagonists of PGE2 receptor (EP1/EP2/EP4) suggests that the suppressor effect of this prostanoid on Th17 cell differentiation is mainly mediated by EP4/cAMP/PKA activation pathway. Taken all together, our results show that PGE2 is able to suppress Th17 differentiation in vitro through EP4 signaling. Further studies are needed to clarify how PGE2 can modulate the Th17 differentiation using in vivo model.
Financial Support: FAPEP 14/17374-3, 12/23580-0, 11/17611-7.
Chronic antigen-specific T helper 17 inflammatory responses in the lungs require persistent adjuvant exposure to be sustained
Authors: Shalaby Karim H , Whitehead Gregory S. , Thomas Seddon Y. , Cook Donald N. 
Published: January 2023

Abstract
Allergic asthma is characterized by exaggerated lung T helper 2 (Th2) and Th17 responses which require signals that stimulate the innate immune system, provided by exposure to environmental adjuvants with an allergen. Once airway disease has developed, it is unclear as to whether the same mechanisms that initiated the disease remain important in sustaining it. We investigated the contribution of persistent innate danger signals in sustaining a chronic antigen-specific Th17 cell response. Animals were sensitized and challenged acutely by aspiration of ovalbumin (OVA) with different adjuvants, including house dust extract (HDE). They were then challenged for a further 5 weeks with OVA alone or OVA + adjuvant to test whether the adjuvant becomes redundant in sustaining airway inflammation once it has already been established. Removal of the adjuvant during the chronic challenges resulted in the animals displaying minimal airway inflammation, whereas continuous challenge with adjuvant and OVA caused significant inflammation. HDE was particularly potent at promoting airway neutrophilia, lymphocytosis and greater IL-17A production from lung explants restimulated ex vivo with OVA, suggesting that persistent exposure to this adjuvant amplified the antigen-specific Th17 response. Furthermore, CD4+ cells from OVA-receptor transgenic (OT-II) IL-17A-fate mapping mice were differentiated under Th17 polarizing conditions and adoptively transferred to recipients which were then challenged with either OVA alone, OVA + HDE or HDE alone. Persistent exposure to HDE and OVA, compared to either one alone, amplified the lung antigen-specific memory Th17 response, independently of the enzymatic activity of HDE but rather via TLR4 signaling.
Characterization of antigen-specific CD8 T cell responses in experimental cerebral malaria
Authors: Urban Stina Lisa , Pewe Lecia L , Harty John T 
Published: January 2023

Abstract
Malaria remains a major global health concern, infecting more than 200 million individuals and resulting in almost a half a million deaths per year. Most malaria-associated deaths are caused by cerebral malaria (CM). Due to ethical limitations of human experimentation, an experimental cerebral malaria (ECM) model is used to further our understanding of the pathogenesis of CM. C57BL/6 mice are susceptible to ECM upon Plasmodium berghei ANKA live sporozoite (spz) or infected RBC (iRBC) infection. CD8 T cells are required for ECM symptoms as depletion of CD8 T cells inhibits disease. Despite the known role of CD8 T cells in the pathogenesis of ECM, the kinetics, phenotype and function of antigen-specific response have not been described. Here, we characterize the antigen-specific CD8 T cell phenotype after spz or iRBC infection and correlate with parasitemia levels and development of ECM. Using surrogate activation markers to identify antigen-specific CD8 T cells, the frequency of CXCR3 expressing activated CD8 T cells decreased initially and subsequently increased during onset of ECM symptoms. Higher parasitemia levels and frequency of activated CD8 T cells correlated with ECM development in both spz and iRBC infection models. Based on these data, we hypothesize that ECM develops in a goldilocks-principle manner. If a specific threshold of parasitemia and activated CD8 T cells are reached at the same time, ECM develops, but should the CD8 T cell response occur too early or late, ECM does not develop and mice will eventually succumb to hyperparasitemia and severe anemia.