bluuebunny/crossref_metadata_2025 huggingface dataset:
HUMAN LYMPH NODE DERIVED FIBROBLASTIC RETICULAR CELLS AND THEIR CHEMOKINE EXPRESSION PROFILE AFTER AN INFLAMMATORY STIMULUS
Authors: Marti Luciana C , Palomino Diana Torres , Almeida Camila Bononi , Pasqualim Denise Cunha , Cury Adriano , de Oliveira Salvalaggio Paolo Rogério , Macedo Antonio Luiz , Severino Patricia , Rizzo Luiz Vicente
Published: January 2023

Lymph node is a secondary lymphoid organ with highly organized and compartmentalized structure. Fibroblastic reticular cells (FRC) integrate LN structure, and are characterized by podoplanin expression (PDPN) and lack of CD31. FRCs are involved in several immune response processes but the mechanism is still under investigation. Another cell population present in the lymph node is the one lacking PDPN and CD31, so called, double negative population (DNC). FRCs and DNCs are described as chemokines producers, controlling migratory pattern and positioning of immune cells during homeostasis or inflammation. We evaluate the gene expression of human FRCs and DNCs after an inflammatory stimulus. Lymph nodes were obtained from 04 patients with different clinical diagnosis submitted to surgical procedures. All patients signed an informed consent (Approved by Ethic Committee - CAAE: 07768712.4.0000.0071). All lymph nodes were analyzed by histopathology, they were also enzymatic digested. Cells were characterized and sorted in two populations FRCs and DNCs. These cells were maintained without stimuli or stimulated with TNFα + IL-1β for 24hours. Next, RNA was extracted; gene expression was characterized using DNA Microarray. Our results demonstrate up-regulation of chemokines such as CCL2, CCL3, CCL5, CCL7 CXCL1, CXCL2, CXCL8 and CXL10 (p<0.05) in both cells subsets after inflammatory stimuli, the secretion enhancement of CCL2, CCL20 and CXCL8 was confirmed by ELISA. The up-regulated expression of CCL3L3, CCL11, CCL13, CXCL5, CXCL6 only by FRCs and CCL8, CXCL3 only by DNCs, may link these cells to additional properties that need to be further investigated. These results show that FRC and DNC positively respond to an inflammatory stimulus.
Unexpected renal tissue protective role for IL-17 following disseminated candidiasis
Authors: Biswas Partha Sarathi , Ramani Kritika , Garg Abhishek V. , Conti Heather R , Whibley Natasha , Kolls Jay K , Gaffen Sarah L
Published: January 2023

The incidence of life-threatening disseminated Candida albicans infections is increasing in hospitalized patients, with fatalities as high as 60%. Death from disseminated candidiasis in a significant percentage of cases is due to fungal invasion of the kidney, leading to renal failure. Treatment of candidiasis is hampered by drug toxicity, the emergence of anti-fungal drug resistance and lack of vaccines against fungal pathogens. The cytokine IL-17 is known to be a key mediator of defense against candidiasis. However, the underlying mechanisms of IL-17-mediated renal immunity are poorly understood, and have to date assumed to be solely through the regulation of antimicrobial mechanisms through neutrophils. Here, we identify an unexpected role for IL-17 in inducing the Kallikrein (Klk)-Kinin System (KKS) in C. albicans-infected kidney, which we show to be a major mechanism of renal protection in this disease. Klk1 expression was upregulated in the infected kidney in an IL-17R-dependent manner. Overexpression of Klk1 or treatment with bradykinin rescued IL-17RA−/−mice from candidiasis. Therapeutic manipulation of IL-17-KKS pathways restored renal function and prolonged survival by preventing apoptosis of renal cells following C. albicans infection. Furthermore, combining a minimally effective dose of fluconazole with bradykinin markedly improved survival following candidiasis in comparison to fluconazole or bradykinin alone. These results indicate that IL-17 both limits fungal growth and prevents renal tissue damage in disseminated candidiasis via the KKS. Since drugs targeting the KKS are approved clinically, these findings offer potential avenues for treatment of this fatal nosocomial infection.
Th17/TfH cells in rheumatoid arthritis: correlations with disease activity and therapy response
Authors: McGeachy Mandy J , Singh Deepika , Henkel Matthew , Moreland Larry
Published: January 2023

Th17 and TfH cells are thought to promote tissue inflammation (Th17) and autoantibody production (TfH) in rheumatoid arthritis (RA) and other autoimmune diseases. TfH cells that co-express Th17 markers (Th17/TfH) correlate with disease activity in juvenile dermatomyositis. However, Th17/TfH cells have not been detailed in RA subjects in context of therapy response. Blood T cell and B cell subsets were analyzed by flow cytometry in 16 subjects with active RA, before and after TNF inhibitor therapy. Proportions of CXCR5+ TfH cells that co-expressed the Th17 marker CCR6 (Th17/TfH cells) directly correlated with disease activity at baseline. In contrast, there were no correlations between disease activity and other T cell subsets including total Th17 (CCR6+), TfH (CXCR5+) or Th1 (CXCR3+) cells, or with CXCR3-expressing TfH cells. Th17/TfH cell frequencies were unchanged by TNF inhibition, fitting with no effect of TNF on stimulation of IL-17 or IL-21 in vitro. In fact, Th17/TfH cell proportions showed remarkable stability within individuals over time despite changes in disease activity. Activated class-switched B cells were increased in subjects with high autoantibody, but B cell subsets did not correlate with disease activity or with Th17/TfH cells. Finally, baseline Th17/TfH cells correlated with numbers of swollen joints at baseline and following one year of stable therapy. These data demonstrate the stability of chemokine receptors as markers of blood T cell phenotypes within individuals, and point to differences in underlying T cell phenotypes as drivers of autoimmune joint disease activity and response to TNF inhibitor therapy.
Potential anti-nociceptive role of chemokine CXCL1 in peripheral nerve injury-induced neuropathic pain
Authors: Malon Jennifer T , Cao Ling
Published: January 2023

Our previous studies have indicated that both microglial CD40 and spinal cord infiltrating CD4+ T cells play critical roles in the maintenance of peripheral nerve injury-induced neuropathic pain using a murine model of neuropathic pain, spinal nerve L5 transection (L5Tx). It is well accepted that chemokines play important role in the development of neuropathic pain. To further understand the role of microglial CD40 and CD4+ T cells in peripheral nerve injury-induced spinal cord chemokine response, we determined the levels of various chemokines in the lumbar spinal cord in CD40 knockout (KO), CD4 KO, and wild type (WT) BALB/c mice post-L5Tx. Using a multiplex assay, a total of eight chemokines CXCL1 (KC), CCL1 (TCA-3), CCL2 (MCP-1), CCL3 (MIP-1 alpha), CCL5 (RANTES), CCL11 (Eotaxin), CCL17 (TARC), and CCL22 (MDC) were examined. In WT mice, but not KO mice, L5Tx induced significant, gradual increases in CCL2, CCL3 and CCL5 up to day 21 post-L5Tx. At RNA level, L5Tx induced less pronounced increases of these chemokines in both types of KO mice compared to WT mice. These reduced chemokine response correlate with our previous observation that both CD40 KO and CD4 KO mice displayed significantly less mechanical hypersensitivity following L5Tx. Interestingly, L5Tx induced a significant increase in CXCL1 only in CD40 KO mice. Intrathecal injection of CXCL1 significantly reduced L5Tx-induced mechanical hypersensitivity in WT mice, indicating a potential protective effect of CXCL1 in neuropathic pain. Altogether, our data suggest chemokines’ roles in the development of neuropathic pain are chemokine type-specific.
NOD1 ligand administration restores optimal steady-state hematopoiesis in germ-free mice
Authors: Iwamura Chiaki , Jankovic Dragana , Bouladoux Nicolas , Belkaid Yasmine , Sher Alan
Published: January 2023

The microbiota is required for optimal hematopoiesis. When compared to specific-pathogen free (SPF) mice, germ free (GF) animals display decreased numbers of hematopoietic stem cells (HSC), common myeloid progenitors (CMP) and common lymphoid progenitors (CLP) in bone marrow. Muropeptides produced by both gram-negative and gram-positive bacteria are important agonists of the innate immune response and are recognized by the intracellular pattern-recognition receptors NOD1 and NOD2. In the present study, we examined both direct and indirect effects of NOD signaling on hematopoietic homeostasis. While stimulation with NOD1 or NOD2 ligands had no effect on survival/proliferation of HSC, CMP and CLP in vitro, exposure of bone marrow-derived stroma cells (BMSC) to NOD1, but not NOD2, ligand induced expression of the differentiation factors SCF, IL-7, IL-3, Thpo and IL-6. Interestingly LPS, which is known to act directly on hematopoietic precursors, selectively induced only IL-6 in BMSC. To test the effects of NOD1 ligand in vivo, we compared groups of GF animals with and without peroral NOD1 ligand administration. NOD1 ligand treatment restored to normal levels serum concentrations of SCF, Thpo and IL-6, as well as the numbers of HSC, CMP and CLP in bone marrow. Taken together, these findings demonstrate that NOD1 ligand induces BMSC to produce cytokines that regulate the size of the major hematopoietic precursor pools. Thus, NOD1 signaling appears to be a major factor underlying the requirement for the microbiota in the maintenance of steady-state hematopoiesis.

This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious diseases.
Compromised emergency hematopoiesis in obesity requires TLR4
Authors: Borghesi Lisa A , Liu Ailing , Ding Ying
Published: January 2023

Following acute injury/infection, bone marrow (BM) precursors must responsively redirect from steady-state to emergency hematopoiesis. Prospective and retrospective studies establish obesity as risk factor for infection but the mechanisms are poorly understood. Here, we show in a murine model of obesity that moderate changes in steady-state BM hematopoiesis become exaggerated during hematopoietic duress. At steady-state, diet-induced obese mice have a 30% decrease in BM hematopoietic stem cells (HSCs) with a parallel increase in splenic HSCs. HSCs are functionally competent in vitro and following competitive transfer in vivo. BM lymphoid and myeloid progenitors in obese mice are numerically intact but the transcriptional profiles and differentiation potential are strikingly myeloid-biased, accompanied by myeloid skewing of peripheral blood. Responses to acute hematopoietic challenge are deficient. Emergency hematopoiesis following chemotherapeutic ablation is reduced 60% in obesity. Likewise, emergency myelopoiesis in response to endotoxin is characterized by accumulation of immature rather than mature myeloid cells in obese mice. The obesity-associated inflammatory environment plays a key role as transfer of obese BM to lean recipients restores emergency immune response to chemotherapeutic challenge. Moreover, mice deficient in toll-like receptor 4 (TLR4), a central regulator of inflammation, retain robust hematopoietic rebound responses despite diet-associated weight gain. Thus, in obesity, moderate changes in steady-state blood cell production mask major defects in hematopoietic stress responses, via a TLR4-dependent mechanism.
Ubiquitylation of the Paf oncoprotein and interaction with PCNA is essential for hematopoietic stem cell function and development.
Authors: Shettigar Megha , Savic Miloje , Sant’Angelo Derek B , Denzin Lisa K
Published: January 2023

Hematopoietic stem cells (HSCs) are critical for the lifelong production and maintenance of all blood cell types. The molecular mechanisms that guide this process remain poorly understood. The 15kDa proliferating cell nuclear antigen (PCNA) associated factor (Paf) is a potent oncogene that is over-expressed in most cancers. We have previously shown that Paf is essential for HSC and progenitor function and development. Paf deficient mice (Paf−/−) are leukopenic due to reduced number of HSCs and committed progenitors. Paf−/− HSCs failed to maintain quiescence, to self-renew and to support long term hematopoietic reconstitution. To determine the in vivo molecular interactions and pathways by which Paf functions to mediate hematopoiesis, we introduced mutant versions of Paf into the Paf−/− mice. These unique mouse models allowed us to show that Paf-PCNA interactions and Paf ubiquitylation are both essential for hematopoiesis. Furthermore, biochemical analyses of cells from these mice showed that Paf interaction with PCNA was essential for nuclear localization and proper ubiquitylation of Paf. Collectively, therefore, our studies show that Paf function is dependent upon the ability to interact with PCNA and that the ubiquitylation of Paf regulates Paf’s function during hematopoiesis. Analyses are ongoing to further delineate the molecular mechanism by which Paf mediates HSC function and development.
T cell targets in mouse allergy extend beyond the major mouse allergen Mus m 1
Authors: Schulten Véronique MJ , Westernberg Luise , Paul Sinu , Sette Alessandro
Published: January 2023

Mouse allergy is an important disease affecting laboratory workers and inner-city households with increased exposure leading to allergic sensitization and asthma. To date, only one major allergen in mouse, Mus m 1, has been identified. Using a combination of proteomics and bioinformatics, we sought to identify T cell targets in mouse allergic patients.

Mouse urine and epithelial extract were analyzed by 2D- IgE/IgG immunoblots using pooled sera from mouse allergic donors. Mass spectrometry of selected protein spots identified 30 novel antibody reactive proteins. Predicted MHC binding peptides from these novel proteins and mouse homologs to mammalian allergens were screened for T cell reactivity in PBMCs from mouse allergic patients. Overlapping peptides from the major mouse allergen Mus m 1 were screened in parallel.

T cell epitope mapping experiments that responses to Mus m 1 peptides were dominant overall. However, reactivity to mouse peptides homologous to other mammalian allergens was also detected. Very little T cell reactivity against peptides derived from novel proteins was observed.

In summary, our data demonstrates that the cellular and serological targets of the allergic response overlap, with Mus m 1 being the major target for both T cells and IgE antibodies. However, on a serological level, other proteins are also recognized. Furthermore, mammalian allergens with murine homologs may also be of relevance for the T cell response in mouse allergic patients. Identifying the immunological targets in mouse allergy is of high importance since the prevalence of this disease is drastically increasing and candidates for improved diagnostics and immunotherapeutic approached are of growing importance.
Toll like receptor 2 co-stimulation signaling in CD4+ T cells promote TH9 differentiation and function
Authors: Karim Ahmad Faisal , Reba Scott M , Ding Xuedong , Rojas Roxana E
Published: January 2023

We have recently demonstrated that mycobacterial ligands engage Toll like receptor 2 (TLR2) on human CD4+ T cells and up-regulate T-cell receptor (TCR) triggered-Th1 responses in vitro and in vivo. To better understand the role of T-cell expressed TLR2 on CD4+ T cell differentiation and function, we conducted a gene expression analysis of naïve CD4+ T-cells stimulated in the presence or absence of TLR2 co-stimulation. Unexpectedly, naïve CD4+ T-cells co-stimulated via TLR2 show a significant up-regulation of IL-9 mRNA compared to cells co-stimulated via CD28. To further evaluate the impact of T-cell expressed TLR2 on TH9 differentiation, naïve CD4+ T cells were polyclonally stimulated under TH9 polarizing conditions with or without a TLR2 agonist. Upregulation of TH9 differentiation, evidenced by increases in both percent of IL-9 secreting cells and IL-9 in culture supernatants, was observed in cells differentiated in the presence of TLR2 agonist. Also, engagement of T-cell TLR2 up-regulated the percent of IL-9 secreting cells in Ag85B-specific CD4+ T-cells stimulated with cognate peptide in TH9 polarizing conditions. On the other hand, in the absence of TH9-polarizing conditions, TLR2 engagement on CD4+ T-cells had minimal effect on IL-9 secretion and TH9 differentiation. This is likely due to a prominent effect of TLR2 signaling on IFN-γ secretion and TH1 differentiation under non-polarizing conditions. Here we also report that, TLR2 signaling in CD4+ T-cells increased expression of transcription factors BATF and PU.1, which are known to positively regulate TH9 differentiation. These results reveal a novel role of T-cell expressed TLR2 in enhancing the differentiation and function of TH9 T cells.
Activated MLKL functions as signal II of the NLRP3 inflammasome
Authors: Gutierrez Kimberley Diane , Davis Michael , Olsen Tayla M , Oberst Andrew
Published: January 2023

Activation of the NLRP3 inflammasome results in caspase-1 dependent processing and secretion of IL-1β. Inflammasome activation and IL-1β maturation is thought to require two signals: Signal I leads to transcriptional up-regulation of inflammasome components and pro-IL-1β. Various agents such as ATP and nigericin can function as signal II to activate the NLRP3 inflammasome through a mechanism that is thought to involve disrupted ion homeostasis. This in turn leads to IL-1β processing and release, as well as pyroptotic cell death. Notably, the execution of another cell death program, necroptosis, also involves cellular membrane disruption and ion flux, through the action of the protein MLKL. Here, we demonstrate that the execution of necroptosis by MLKL can lead to NLRP3 inflammasome activation and the secretion of bioactive IL-1β. We further show that IL-1β processing is independent from the cell death function of MLKL. This mechanism represents a novel inflammatory signal emanating from necroptotic cells, as well as an unexpected mechanistic cross-talk between necroptosis and inflammasome activation.
The Role of Glutathione Reductase in Neutrophil Respiratory Burst and ERK Signaling
Authors: Liu Yusen , Crowell Sara A. , Chen Ying , Rogers Lynetter K. , Nelin Leif D.
Published: January 2023

Respiratory burst plays a crucial role in the killing of bacteria by phagocytes. During the respiratory burst, NADPH oxidase complexes are assembled on the membrane to produce superoxide, which is then converted to H2O2 for pathogen killing. Glutathione (GSH) is crucial for the detoxification of H2O2 that has diffused into the cytosol. GSH is generated via both de novo synthesis and regeneration from glutathione disulfide. The rate-limiting step in GSH synthesis is catalyzed by γ-glutamate cysteine ligase, composed of a catalytic subunit (Gclc) and a regulatory subunit (Gclm), whereas GSH regeneration is catalyzed by glutathione reductase (Gsr). Mice with a mutation in the Gsr gene exhibit a profound defect in immune defense against bacteria, which is associated with compromised respiratory burst activity. To address the mechanisms underlying the compromised respiratory burst, we purified neutrophils from wildtype (WT) and Gsr−/− and Gclm−/− mice, and assessed their respiratory burst in response to phorbol ester (PMA). Compared to WT neutrophils, Gsr−/− neutrophils exhibited a dramatic decrease in respiratory burst activity. A similar trend was observed in neutrophils derived from Gclm−/− mice, which have 10–40% of normal tissue GSH levels. However, no significant defect in immune defense against E. coli was found in Gclm−/− mice. Cellular fractionation indicated that the NADPH oxidase subunits underwent membrane translocation in response to PMA in both WT and Gsr−/− neutrophils. Interestingly, Gsr−/− neutrophils also displayed a dramatic enhancement in activation of the ERK MAP kinase pathway. Our results indicate that Gsr plays an important role in the regulation of both the respiratory burst and cell signaling in neutrophils.
Macrophages and Plasmacytoid Dendritic Cells Initiate Early Immune Response To Severe Malaria In The Bone Marrow
Authors: Lauvau Gregoire , Spaulding Emily , Fooksman David R , Reizis Boris , Daily Johanna P
Published: January 2023

Malaria still remains a significant global health problem worldwide with approximately 200 million people infected, and half a million deaths every year. Most of these fatalities (80%) occur in children under the age of five, and the latest RTS, S, preventative vaccine trial only exhibited low protective efficacy (below 30%). The blood stage of Plasmodium infection accounts for the clinical symptoms of malaria, yet the immune mechanisms underlying disease outcomes are poorly understood. Here we have analyzed a cohort of severely infected Malawian children and revealed dramatic levels of inflammatory cytokines and extremely robust blood leukocyte activation, notably for CD14+CD16lo CCR2+ inflammatory monocytes, plasmacytoid DCs (pDCs), NK and T cells. We could recapitulate these observations using the Plasmodium yoelii (Py) 17X YM surrogate mouse model of lethal blood stage malaria. Using this experimental system, we demonstrate that type I interferon (IFN) signals as a key cytokine controlling lethal outcomes and immune cell activation. Plasmacytoid dendritic cells represented the main cellular source of type I IFN in the bone marrow and the blood of infected mice. Most interestingly, the activation of pDCs required CD169+ macrophages, and both cell types exhibited prolonged interactions in the bone marrow of infected mice by intravital microscopy. We will present additional data uncovering the molecular sensing pathways controlling these processes. Altogether our study establishes essential molecular pathways and cellular interactions that occur during this significant human parasitic infection, and suggest novel potential therapeutic targets involved in severe malaria.
Alternatively activated brain-resident macrophages acquire and retain inflammatory properties following CNS infection while interacting with effector and memory T cells.
Authors: Rua Rejane , McGavern Dorian B
Published: January 2023

The meningeal and perivascular spaces of the central nervous system (CNS) are inhabited by specialized macrophages, but their homeostatic status and role in orchestrating the immune response against invading pathogens is not well understood. Examination of the naive brain by two-photon microscopy revealed that meningeal and perivascular macrophages are highly dynamic and constantly survey their immediate surroundings similar to microglia. Under steady state conditions, we uncovered that they are maintained in an alternatively activated state, which likely facilitates brain homeostasis. Interestingly, during the development of fatal meningitis induced by lymphocytic choriomeningitis virus, these cells were directly engaged by infiltrating virus-specific CD8+ T cells following acquisition of viral antigen and conversion into an inflammatory phenotype. Mechanistically, microarray analyses revealed that this transition relied on innate cytokine signaling and occurred in the absence of infiltrating inflammatory cells. Using a sub-lethal model of viral meningitis, we observed that despite elimination of previously infected cells, CNS macrophages remained activated for weeks after viral clearance, which was dependent on IFN-g signaling and associated with tissue-resident memory T cell interactions. Collectively, these data indicate that brain-resident macrophages are highly plastic cells that can quickly participate in the antiviral defense against an invading pathogen and can also become imprinted with a prolonged activation program. The inflammatory properties and localization of these cells may explain why most CNS immune responses first develop in the meningeal and perivascular spaces.
Unmasking two stages of interferon signaling in dendritic cells infected with HIV-1
Authors: Johnson Jarrod S , Lucas Sasha , Skelton Stephanie , Nazitto Rodolfo , Amon Lynn , Littman Dan R , Aderem Alan
Published: January 2023

Myeloid dendritic cells (DCs) have the innate capacity to sense pathogens and mount counteroffensive responses, which include the production of type I and type III interferons (IFN). In the case of HIV-1 infection, DCs can internalize and present viral antigens, but the majority of DCs are not productively infected due to restriction at the level of reverse transcription. Consequently, DCs do not efficiently ‘sense’ HIV-1, fail to produce IFN, do not mature, and cannot program appropriate adaptive immune responses. To improve our understanding of how IFN and cell maturation are regulated in monocyte-derived DCs, we have used systems analyses to study how DCs respond to HIV-1 after removing the block to reverse transcription with the lentiviral protein Vpx. We have profiled changes in open chromatin during infection using an assay for transposase-accessible chromatin (ATAC-seq), and have tracked the kinetics of gene expression in parallel. Using antiretroviral drugs and virus mutants to separate stages of the life cycle, we have determined that at least two signals cooperate to drive innate immune responses during HIV-1 infection: the first coming from the cytoplasmic sensor, cGAS, which is known to recognize reverse transcribed HIV cDNA, and the second from an unrelated pathway engaged after virus integration. Moreover, by interrogating infection in combination with a battery of classic innate agonists we have found that HIV-1 primes DCs to produce IFN, even in the presence of certain antiretroviral drugs. Our studies demonstrate that robust IFN production in response to HIV-1 infection depends on the coordinated action of distinct transcription factors and illuminate key pathways that regulate innate immunity in myeloid cells.
The impact of surgical wounding on pulmonary infection
Authors: Xu Yun , Crane Meredith J. , Henry William L. , Albina Jorge E. , Jamieson Amanda M.
Published: January 2023

Post-surgical pneumonia happens frequently in hospitals and is prevalent in intensive care unit (ICU) patients. Its prevalence is correlated with the length of stay in hospital, the degree of trauma, and importantly, the mortality. While it is clear that there is an increased risk of acquiring pneumonia in post-surgical patients, not much is known about the detailed impacts of surgical wound healing on pulmonary infections and its immunity. Therefore, it is necessary to understand why pneumonia is common after surgery, and how the wound healing process influences the lung. Wound healing is a complicated process involving various immune cells, such as neutrophils, monocytes, and macrophages. Immune cells essential for proper wound healing are also crucial for the host to recognize, fight and resolve pulmonary infection. We have combined two wounding models (dorsal polyvinyl alcohol sponge implantation and excisional tail wounding) with one pulmonary pathogen—Influenza A Virus (IAV)—to investigate the immune responses at the two distinct sites. From our investigation, we have found that 1) wounding does not influence the clearance of IAV; 2) wounding increases immune cell infiltration into the lung; 3) wounding increases the levels of cytokines and chemokines in the lung; and 4) IAV infection delays wound healing. The future direction of this project will focus on investigating how the changes in immune cell infiltration, cytokines, and chemokines happen, and what renders the host more susceptible to pulmonary infections. In addition, we will explore the interactions between wounding and pulmonary Streptococcus pneumoniae infection and the general biological mechanisms behind the clinical phenomena.
NLRP12 limits TNFα-induced apoptosis of monocyte progenitor cells during emergency hematopoiesis
Authors: Linz Brandon M , Brickey W June , Ting Jenny P , Cairns Bruce A , Maile Robert
Published: January 2023

The NLR family member NLRP12, expressed in peripheral leukocytes and bone marrow progenitor cells, is implicated in controlling the response of the innate immune system by suppressing non-canonical NfκB signaling. Given the hematopoietic system’s ability to respond to insults by rapidly increasing myeloid cell production, and the importance of tightly controlled cytokine regulation during these processes, we hypothesized NLRP12 to be a key regulator of the innate immune system’s reconstitution following a hematopoietic emergency. To test this, wild type and Nlrp12−/− C57BL/6 female mice underwent a radiation-thermal combined injury (RCI) consisting of a 20% total body surface area, full thickness contact burn in addition to receiving a single whole-body dose of 5 Gy γ-radiation. Following injury, Nrlp12−/− mice showed a three-fold increase in specific apoptosis of granulocyte-monocyte progenitor cells leading to a reduction in the total number of bone marrow cells and thus systemic leukopenia. We also observed that the lack of NLRP12 led to elevated levels of TNFα systemically following injury. Utilization of a neutralizing antibody against TNFα following injury abrogated the granulocyte-monocyte progenitor cell apoptosis and leukopenia seen in vehicle-treated mice. These data lead us to conclude that NLRP12 acts as a suppressor of TNFα signaling during emergency hematopoiesis; a novel finding, since no member of the NLR family has been previously found to interact with TNFα signaling.
Inflammasome dependent IL-1β secretion requires Dnase1L3 activity
Authors: shi guilan , Abbott Kennady , Salter Russell D , Keyel Peter A
Published: January 2023

Inflammation is driven by inflammatory cell death and the secretion of pro-inflammatory cytokines like IL-1β and alarmins like High Mobility Group Box Protein 1 (HMGB1) by the inflammasome. The inflammasome is a cytosolic multiprotein complex that typically contains a Nod-like receptor (NLR), like NLRP3 or NLRC4, and the adaptor ASC which links to caspase-1 for activation. How signals from the cytoplasmic NLR induce nuclear ASC and HMGB1 translocation into the cytosol for inflammasome function remains elusive. Here we hypothesize that Dnase1L3 (DNase γ), a Ca2+/Mg2+-dependent endonuclease, facilitates inflammasome-mediated release of cytokines and alarmins. When we treated murine macrophages with the Dnase1L3 inhibitors Fmoc-D-cyclohexylalanine (FCA) or pontacyl violet 6R (PV), HMGB1 and IL-1β release were inhibited following NLRP3 or NLRC4 stimulation. FCA and PV did not directly inhibit Casp1. We confirmed the specificity of the inhibitors using RNAi silencing of Dnase1L3 in THP-1 cell lines. Interestingly, pyroptosis was only slightly blocked by Dnase1L3 inhibition. Mechanistically, we found that ASC nuclear translocation and speck formation was impaired following FCA treatment, as was Casp1 cleavage. These data suggest that Dnase1L3 inhibition prevents Asc release from the nucleus. This demonstrates that pyroptosis and cytokine release can be mechanistically separated. Taken all together, these data show that Dnase1L3 is necessary for inflammasome-mediated release of cytokines and alarmins.
Hepatitis C virus hijacks host microRNAs to dampen antiviral interferon signaling
Authors: Jarret Abigail P , McFarland Adelle P , Horner Stacy M , Kell Alison M , Hong MeeAe , Badil Samantha , Joslyn Rochelle C , Baker Darren , Carrington Mary , Hagedorn Curt , Gale Michael J , Savan Ram
Published: January 2023

Hepatitis C virus (HCV) is a hepatotrophic ssRNA virus that infects over 160 million people worldwide and 60–80% of HCV cases will persist to a chronic infection, a serious risk factor for liver diseases. We recently discovered that HCV infection of hepatocytes induces expression of two microRNAs (miRNAs), miR-208b and miR-499a-5p. We found that these miRNA are capable of decreasing IFNL2 and IFNL3 expression to aid viral replication. Here we show that miR-208b and miR-499a-5p block type I IFN signaling in HCV-infected hepatocytes by directly targeting and down regulating the interferon (alpha, beta and omega) receptor 1 (IFNAR1) mRNA. Inhibition of these miRNAs during HCV infection of hepatoma cells rescued expression of IFNAR1 and improved the antiviral response to exogenous type I IFN. Interestingly, prolonged treatment of HCV-infected hepatocytes with type I IFNs led to a synergistic induction of miR-208b and miR-499a-5p, suggesting a critical role for type I IFN in the regulation of these miRNAs. As miR-208b and miR-499a-5p can target and suppress the IFNL3 risk allele, it is possible that increased miRNA expression following IFN treatment may partly explain why this genotype associates with poor response to IFN based therapies. Collectively these data define a new role for miR-208b and miR-499a-5p as HCV-induced suppressors of type I IFN signaling and add to our understanding of HCV immune evading strategies.
Cognitive impairment, blood-brain barrier breakdown, and pro-inflammatory mediators in neonatal Escherichia coli K1 meningitis.
Authors: Barichello Tatiana , Generoso Jaqueline , Simoes Lutiana , Florentino Driélly , Michels Monique , Comim Clarissa M , Petronilho Fabricia , Quevedo Joao , Dal-Pizzol Felipe , Teixeira Antonio
Published: January 2023

Neonatal Escherichia coli meningitis is an important cause of mortality and morbidity in newborns. We evaluated cytokines, oxidative damage/enzymatic defense, BDNF levels, blood-brain barrier (BBB) integrity in the hippocampus of neonatal Wistar rats following E. coli K1 meningitis and subsequent behavioral parameters in adulthood. Neonatal male rats received an intracisternal injection of placebo or E. coli K1 suspension. First: at 6, 12, 24, 48, and 96 h were evaluated cytokines, oxidative damage/enzymatic defense, BDNF levels, and BBB integrity. TNF-α, IL-4, IL-6, IL-10, BDNF, and protein carbonylation were produced mainly in the first 6 to 96 h. Myeloperoxidase, thiobarbituric acid reactive-substances, nitrate/nitrite, and sulfhydryl group were produced at 24 h. Superoxide dismutase decrease in the first 6 h, catalase activity levels did not change and the BBB breakdown at 12 h after meningitis induction. Second: sixty days after meningitis were evaluated behavioural parameters (neonatal rats received antibiotic treatment): habituation to the open-field, step-down inhibitory avoidance, and continuous multiple-trials step down inhibitory avoidance tasks. In the habituation to the open-field and in the step-down inhibitory avoidance tasks there were no differences between training and test session in the meningitis group, demonstrating memory impairment. In the continuous multiple-trials step-down inhibitory avoidance task, there was a significant increase in the number of training trials required to reach the acquisition criterion in the meningitis group when compared to the control group, indicating a learning impairment. Neonatal E. coli K1 meningitis causes learning and memory impairments in adulthood.
Estimating total numbers of Mycobacterium tuberculosis in lymph nodes of cynomolgus and rhesus macaques
Authors: Ganchua Sharie Keanne C , Cadena Anthony M , Flynn JoAnne L
Published: January 2023

Tuberculosis (TB) is predominantly thought of as a lung disease. However, in classic pathology studies, a thoracic lymph node is often infected in addition to the lungs. Furthermore, lymphadenitis is common in children infected with Mycobacterium tuberculosis (Mtb). In our non-human primate model, at least one lymph node is infected in nearly all macaques. However, rhesus macaques have substantially more lymph node involvement than cynomolgus macaques, and this often leads to clinical deterioration. To better understand the role of lymph nodes in TB, as well as the differences between these species, we quantified the total number of Mtb genomes (live and dead bacteria) in the lymph nodes at 4 and 12 weeks post-infection (p.i.). Mtb burden in the lymph nodes was not significantly different between cynomolgus and rhesus macaques, as well as between 4wk and 12wk p.i.. We then compared the number of Mtb genomes with the number of viable bacteria to determine the degree of bacterial killing in these tissues. Overall, bacterial killing in lymph nodes was very low, compared to lung granulomas. However, the 12wk p.i. rhesus macaques had significantly less Mtb killing in their lymph nodes than the 4wk p.i. rhesus and 12wk p.i. cynomolgus macaques, which could in part explain the greater lymph node involvement in rhesus macaques. The level of bacterial killing between intact and effaced (necrotic) lymph nodes was similar; however, there was a trend of increasing Mtb burden as the effacement increases. Some lymph nodes without culturable bacteria were found to have Mtb genomes present, suggesting lymphatic spread. Our preliminary data suggest that lymph nodes may play a role as a bacterial reservoir and as a means of bacterial dissemination and reactivation.
M. tuberculosis Mce3C promotes mycobacterial mammalian cell entrance and enhances mycobactrial intracellular survival.
Authors: Liu Cui Hua , Li Jie , Li Bingxi , Wang Jing
Published: January 2023

It has been suggested that the mammalian cell entry (Mce) proteins are involved in the macrophage cell entry process and the subsequent intracellular survival of Mycobacterium tuberculosis during infection. However, relatively little is known about the specific roles of Mce3C in modulation of phagocytosis and host innate immune defenses. Here we demonstrate that Mce3C can promote the entry of mycobacteria into macrophage cells in a RGD motif-dependent manner. During our attempt to screen for potential Mce3C-interacting host proteins utilizing yeast two-hybrid assay, we found that Mce3C interacts with integrin β2 subunit, which renders it very likely that Mce3C binds β2 family of leukocyte integrins including αLβ2,αMβ2 and αXβ2 through its RGD motif, thus enhancing phagocytosis of mycobacteria by macrophage cells. Furthermore, Mce3C blocks the activation of ERK1/2 signaling pathway, leading to the suppression of Tnf and Il-1β expression, and the promotion of mycobacterial survival within macrophages. Finally, we discover that Mce3C undermines apoptosis of macrophage cells during the course of mycobacterial infection, which further facilitates the intracellular survival of mycobacteria. Thus, these results indicate that M. tuberculosis Mce3C not only strengthens the internalization of macrophage cells by mycobacteria via RGD-mediated interaction with β2 family of leukocyte integrins, but also enhances the intracellular survival of mycobacteria through inhibition of ERK1/2 signaling pathway and apoptosis in macrophage cells.
Recent Thymic Emigrants and Chronic Salmonellosis: How New T Cells Combat an Old Infection
Authors: Goggins J. Alan , Kurtz Jonathan R. , McLachlan James B
Published: January 2023

Typhoid fever, caused by infection with Salmonella bacteria affects approximately 21 million people annually, killing nearly 200,000 and yet the immunological response to this infection is poorly understood. Recent thymic emigrants (RTEs) are the youngest subset of peripheral T cells, which are known to differ in how they functionally contribute to the naïve T cell pool. This distinctive cell population is known to contribute to the maintenance of T cell receptor diversity in the peripheral T cell pool, but their involvement in combating chronic bacterial infections, such as typhoid fever, has not been explored. In the present study, we hypothesized that RTEs are essential contributors to the CD4 T cell response during persistent Salmonella Typhimurium (ST) infection, which predominantly relies on helper T cell immunity to control bacteria. Here, we show that thymectomy of adult mice prior to oral infection with ST leads to increased mortality in addition to significantly increased bacterial burdens in the spleen, mesenteric lymph nodes and liver 30 days post-infection compared to sham surgical controls, suggesting Salmonella-specific RTEs may contribute to bacterial clearance. Surprisingly, thymectomized animals also demonstrated expanded numbers of peripheral Salmonella-specific T cells, compared to control animals, implying that these existing cells are somehow incapable of controlling infection in the absence of RTEs. The results of this study provide insight into how new pathogen-specific T cells leaving the thymus participate in combatting a persistent bacterial infection.
Functional SLC11A1 (NRAMP1) enhances protection from Coxiella burnetii infection
Authors: Hedges Jodi F , Robison Amanda , Kimmel Emily , Jutila Mark A.
Published: January 2023

SLC11A1 is a divalent ion transporter formerly known as the natural resistance-associated macrophage protein (NRAMP1). Also known as the Bcg/Lsh/Ity locus, SLC11A1 was originally characterized as the resistance factor for Mycobacterium, Leshmania, and Salmonella infections. This protein is non-functional in C57BL/6 mice, thus, the susceptibility of this strain to these pathogens. We recently characterized the expression of SLC11A1 in the majority of γδ T cells and NK cells, which are primary sources of innate IFN-γ. Cells expressing SLC11A1 were more prone to activation. We hypothesized that SLC11A1 would have an important function in infections in which IFN-γ was a critical component. Only two genes have been described as potential susceptibility loci for Q fever caused by Coxiella burnetii infection, they are IFN-γ and SLC11A1 in humans. Thus, we assessed C. burnetii infection in congenic mice with and without functional SLC11A1. SLC11A1+ mice had improved disease symptoms, harbored fewer bacteria, and had increased expression of innate cytokines, including IFN-γ. The results indicate that mice expressing functional SLC11A1 were more resistant to infection with C. burnetii, suggesting an expanded role for this protein in innate immune protection.
Interleukin-17 are the major cytokine in chronic gastritis induced by Helicobacter pylori infection.
Authors: Rodrigues Denise Bertulucci , Silva Emerson Wood , Silva Natalia Jacomo , Fonseca Fernanda Machado , Sawan Monica , Ribeiro Betania Maria , Oliveira Adriana Gonçaves , Martins Ruchele Dias Nogueira , Pereira Sanivia Aparecida , Adad Sheila Jorge , Rodrigues Virmondes
Published: January 2023

Helicobacter pylori (H. pylori) is a highly prevalent bacteria directly involved in various diseases of the upper digestive tract such as gastritis, peptic ulcer and gastric cancer. In this study we analyze in situ immune response in gastric biopsies obtained during endoscopic procedures from 49 patients. Quantitative PCR were performed on cDNA copies of total RNA extracted from each biopsies. PCR were done using Taqman system to selected cytokines and cytokines transcriptions factor representative of Th1, Th2, Treg and Th17 lymphocytes. The relative number of mRNA copies were adjusted using the beta actin gene. The analysis were performed on samples grouped according to presence or absence of H. pylori infection. No significant difference was found in the expression of IFN-γ, IL-4 and IL-10 when comparisons were done between patients with and without H. pylori infection. Moreover, no difference was observed in the expression of T-bet, GATA-3 and FoxP3. On the other hand, the levels of IL-17 and ROR-gamma T were significantly higher in samples obtained from patients with H. pylori infection, compared with those from non infected. The results presented here suggest that IL-17 are involved in the chronic inflammatory process induced by H. pylori.
Nano-silver as effective intracellular bactericidal compound in human osteoclasts
Authors: Filgueira Luis , Schwartz Valerie , Kharoubi-Hess Solange , Walch Michael
Published: January 2023

Bone infections are difficult to treat, resulting usually in severe bone destruction. A variety of bacteria can be the cause of bone infections, including staphylococcus aureus. Osteoclasts, which belong to the macrophage family, are the key cells in bone infections, as they are not well equipped for killing bacteria, but rather enhance their bone resorbing function, when activated in the infection environment. In addition, newly recruited blood monocytes differentiate rather towards osteoclasts, instead of becoming effective macrophages. Silver has been known for centuries for its bactericidal activity. This project investigated the bactericidal effects of 80nm nanosilver particles in E. coli infected human osteoclasts, in vitro. Nano-silver was used at 10μg/ml, which was not toxic to the cells or to the bacteria, when applied directly. However, when bacteria-infected osteoclasts were exposed to nano-silver, intracellular bacterial survival was significantly reduced. Interestingly, ceftriaxone did not have any bactericidal effect on intracellular bacteria in osteoclasts. In conclusion, silver compounds should be considered in the future for the treatment of bacterial bone infections.
The role of neutrophils during Orientia infection.
Authors: Shelite Thomas R , Mendell Nicole L , Bouyer Donald H , Walker David Hughes , Soong Lynn
Published: January 2023

Scrub typhus is a seriously neglected disease with approximately one-third of the world’s population at risk of being infected with Orientia tsutsugamushi, and the occurrence of over one million scrub typhus cases annually illustrate its importance in global health. All scrub typhus case studies that report blood cell counts, describe neutrophilia during the course of infection. Patients with confirmed scrub typhus have significant increases in activated neutrophil proteins in serum, and the increase of neutrophil recruiting cytokines. We also observed neutrophilia as well as neutrophil recruitment to infected tissues in intravenously infected mice, suggesting key role for neutrophils in scrub typhus disease progression. To determine the role of neutrophils in this infection, female C57BL/6 mice were lethally challenged, and neutrophils were depleted one day prior to infection (D−1) or six (D+6) days post infection. The effects of neutrophil depletion were observed to be dependent on the time post infection. Animals depleted early (D-1) exhibited more severe pathology at an earlier time point and had disease progression similar to non-depleted animals but had greater survival. Animals depleted 6 dpi recovered weight, and signs of illness had resolved by 12 dpi. Histopathology demonstrated decreased cellular infiltrates when compared to infected, non-depleted animals. Depletion of neutrophils decreased mortality independent of when depleted, but only depletion 6 dpi resulted in amelioration of signs. These data suggest an important role of neutrophils in tissue pathology and disease progression during scrub typhus infection.
Exosomes: A possible mechanism for the development of Chlamydia-induced upper genital tract pathology
Authors: Russell Raedeen Sherilee , Igietseme Joseph U. , Eko Francis O.
Published: January 2023

It is estimated that 70% of chlamydial infections in women are asymptomatic and, if left untreated, can lead to severe irreversible complications including ectopic pregnancy and pelvic inflammatory disease. Chlamydia trachomatis infects cells in the cervix of the female genital tract and ascends to the upper genital tract (UGT) where it can cause diverse pathologies. It has been shown that Chlamydia-induced UGT pathology occurs even in the absence of live Chlamydia in the lower genital tract. The mechanism of ascending infection and disease is largely unknown. We hypothesize that the ascending infection leading to UGT pathology is mediated by exosomes. Exosomes are vesicles that can package intracellular molecules from donor cells and release their cargo into recipient cells thereby manipulating gene expression and cellular activity. To test our hypothesis, HeLa cells were infected with C. trachomatis serovar D and exosomes isolated from culture supernatants were characterized. Cytokines and chemokines from exosomes were assessed by a multiplex cytokine ELISA assay. Furthermore, purified exosomes were co-cultured with uninfected cells and the protein content of the lysed cells was examined. The results showed infected cells released higher concentrations of exosomes during early infection compared to uninfected cells and significantly higher levels of inflammatory cytokines were associated with these exosomes. Moreover, exosome recipient cells were found to contain chlamydial proteins. These results suggest exosomes could transport pathogenic chlamydial proteins from infected to uninfected cells with potential to stimulate inflammation in recipient cells and hence a possible mechanism for development of UGT pathology.