bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Expression of Toll-like Receptor 2 mRNA after Facial Nerve Injury in Rat
Authors: Park Dong Choon , Min Hye Kyu , Lee Jae Min , Kim Young Il , Kim Sang Hoon , Yeo Seung Geun
Published: February 2023

Objectives:

The aim of this study was to determine whether TLRs mRNA is expressed in the distal facial nerve after facial nerve injury.

Methods:

The adult male Sprague-Dawley with a body weight of 200–250g underwent injury of unilateral facial nerve. Crush or cutting injury was given at the midpoint between the facial nerve trunk and the facial nerve branch. The whisker movement of vibrissae muscle was examined 4 and 14 days after facial nerve injury. The normal facial nerve on the left side and the damaged facial nerve on the right side were taken under the inhalation anesthesia and real-time polymerase chain reaction was performed.

Results:

In the whisker movement of vibrissae muscle, the crush group and the cutting group showed worse scores on the 14th day than the 4th scores in each group (p<0.05). On the 4th day of injury, the expression of TLR 7 mRNA was highest in the crush group, and on the 14th day of injury, the expression of TLR 7 mRNA was highest in the cutting group. On the 14th day of injury, the expression of TLR 2 mRNA was significantly higher in the cutting group than in the control group (p<0.05), on the 4th day of injury, the expression of TLR 9, 13 mRNA was significantly lower in the crush and cutting groups than in the control group (p<0.05).

Conclusions:

The expression of TLR 2 mRNA in the distal facial nerve after facial nerve injury in the cutting group was significantly higher than that in the control group, but the expressions of TLR 9 and 13 mRNA in the crush and cutting groups were significantly low. Therefore, TLR mRNA may be involved in facial nerve damage and regeneration.
Tertiary lymphoid organs drive disrupted gut to lymph node communication through association with collecting lymphatic vessels
Authors: Czepielewski Rafael Sanguinetti , Erlich Emma C , Onufer Emily J , Young Shannon , Saunders Brian , Han Yong-Hyun , Wohltmann Mary , Wang Peter L , Kim Ki-Wook , Kumar Shashi , Hsieh Chyi-Song , Yang Ying , Scallan Joshua P , Zinselmeyer Bernd , Davis Michael J , Randolph Gwendalyn J
Published: February 2023

The concept that impaired lymphatic flow during the progression of Crohn’s disease, a major form of inflammatory bowel disease, emerged decades ago, but remains unresolved. In Crohn’s disease, tertiary lymphoid organs (TLOs) are associated with collecting lymphatic vessels (CLVs) of the mesentery that govern lymph outflow from the intestine. Whether TLOs affect lymph transport is unknown. In the TNFDARE mouse model of Crohn’s-like ileitis, TLOs formed at valve regions which supported lymphangiogenic outgrowths to the TLOs. Photoconversion approaches to study cell trafficking indicated that TLOs received immune cells traveling from the intestine but scarcely allowed their egress, effectively trapping DCs, B, and T cells. Using adoptive transfer of microbiota-dependent TCR we found reduced overall Tregs differentiation and their presence shifted from the lymph nodes to TLOs, altering intestinal-induced responses. Strikingly, although anti-TNF therapy dampens gut inflammation, TLO are refractory the therapy. Moreover, passage of soluble fluorescent tracer through TLO-rich CLVs was also impeded, indicating transport defects were intrinsic to the CLVs. Indeed, lymph flow diverted at TLO to neighboring CLV branches, where dysfunctional valves allowed lymph return to the gut wall, accompanied by lymph leakage at the TLOs. Culturing lymphatic endothelial cells with TNF demonstrate that the cytokine alone can reduce key genes found in lymphatic valves. These data indicate that TNF disrupts lymphatic valve maintenance pathways, promoting formation of mesenteric TLOs that broadly impair lymph transit of molecules and cells from the intestine, impairing immune surveillance of the mucosal barrier, altering homeostasis.
Farnesoid X receptor regulates macrophage activation and recruitment to the lung following exposure of mice to nitrogen mustard.
Authors: Banota Tanvi , Murray Alexa , Sowinski Amanda , Kong Bo , Guo Grace L , Laskin Jeffrey D , Laskin Debra L
Published: February 2023

Nitrogen mustard (NM) is a toxic vesicant known to cause acute lung injury which progresses to fibrosis. This is accompanied by the accumulation of pro-inflammatory/cytotoxic M1 macrophages and anti-inflammatory/pro-fibrotic M2 macrophages in the lung. Farnesoid X receptor (FXR) is a bile-acid activated nuclear receptor involved in lipid homeostasis; it has also been shown to exert anti-inflammatory activity. In these studies, we analyzed the role of FXR in regulating macrophage accumulation and activation in the lung following NM exposure. Male and female wild type (WT) and FXR−/− mice were treated with PBS (control) or NM (0.08 mg/kg) via intratracheal instillation. Lung tissue and bronchoalveolar lavage (BAL) fluid were collected 3, 14, and 28 days later. NM caused histopathologic alterations in the lung including inflammatory cell infiltration, septal damage, and epithelial thickening, which were more prominent in FXR−/− mice relative to WT mice at all post exposure time points. This correlated with aggravated increases in BAL protein and cell content. Flow cytometric analysis revealed pro-inflammatory macrophages were most abundant in BAL 3 days post NM, a response more pronounced in FXR−/− mice. In male but not female mice, increases in proinflammatory macrophages persisted in the lung for at least 28 d post NM. In contrast, while anti-inflammatory macrophages also increased in the lung following NM administration, no differences were observed between WT and FXR−/− mice. These findings demonstrate that FXR modulates proinflammatory macrophage responses to NM in a sex-dependent manner and suggest a potential new target for the development of therapeutics to blunt mustard vesicant induced lung inflammation and injury.
NLRP3 inflammasome activation in macrophages is mechanosensitive
Authors: Almgren-Bell Alison , Joshi Hemant , Todd Elizabeth M. , Morley Sharon Celeste
Published: February 2023

Macrophages are innate immune cells that activate the NLRP3 inflammasome to induce inflammatory cytokine IL-1b production and pyroptotic cell death to combat infections. Dysregulated NLRP3 activation escalates inflammatory diseases under mechanical stress, such as ventilator induced injury (VLI), liver fibrosis, acute lung injury (ALI), and idiopathic pulmonary fibrosis (IPF). Macrophages reside in tissues via anchoring to the extracellular matrix (ECM), and change in adhesiveness tunes their inflammatory response via a process known as “mechanotransduction”. However, the underlying mechanism of how mechanical signaling regulates the NLRP3 inflammasome is not fully known. We have found that the NLRP3 inflammasome pathway in macrophages is sensitive to altered stiffness of the culture surface. We cultured bone-marrow derived macrophages (BMDMs) on collagen-coated polyacrylamide gels or silicone gels mimicking physiological tissue stiffness. We observed that NLRP3-induced IL-1b release was smallest on the softest gel (elastic moduli 0.2 kPa) and increased with a stiffer gel (64 kPa). Additionally, macrophages displayed increased adhesiveness and spreading on stiffer gels, indicating enhanced mechanical force. Further examination of how adhesion based-mechanotransduction modulates NLRP3 signaling will help in understanding inflammatory diseases associated with changing adhesive microenvironments such as lung fibrosis. We propose that increased stiffness during lung fibrosis exerts mechanical stress on macrophages and may promote NLRP3 inflammasome activation. Elucidating the mechanosensitivity of the NLRP3 inflammasome will aid in identifying treatment for such inflammatory diseases.
Antigen presentation by CNS-resident microglia and macrophages regulates CD8 T cell infiltration of the brain during central nervous system (CNS) infection
Authors: Goddery Emma , Fain Cori , Lipovsky Chloe , Jin Fang , Hansen Michael , Johnson Aaron J
Published: February 2023

CD8 T cell responses are essential for clearance of infections in the CNS but contribute to neuropathology. T cell interaction with antigen presenting cells (APCs) at CNS barriers is thought to allow activated antigen-specific T cells to infiltrate the CNS during neuroinflammation. However, the role of CNS-resident APCs during CD8 T cell infiltration of the CNS is unclear. To address this, we utilized Theiler’s murine encephalomyelitis virus (TMEV) and Plasmodium berghei ANKA (PbA) to assess the role of APCs during CD8 T cell responses to CNS infection in mice. During infection, we observed robust upregulation of MHC class I on microglia and brain associated macrophages (BAM). Microglia and BAMs were located in close proximity to inflamed vasculature where antigen presentation to T cells could occur. Thus, we hypothesized that microglia and BAMs are critical APCs for CNS infiltration of antigen specific CD8 T cells. To test this, we generated mice with inducible deletion of MHC class I on microglia and BAMs by crossing CX3CR1creERT2 mice with H-2Db or H-2Kb floxed mice (Db cKO or Kb cKO). We found that brain infiltrating virus-specific CD8 T cells were reduced in Db cKO mice compared to controls during TMEV infection, while peripheral priming of T cells was unaffected. Conversely, antigen-specific CD8 T cell infiltration was not impacted in Kb cKO mice infected with TMEV-OVA257–264. In Db cKO mice, brain-infiltrating CD8 T cells were increased during PbA induced experimental cerebral malaria (ECM), indicating a potential inhibitory role for CNS APCs. These results demonstrate that local antigen presentation by microglia/BAMs can both positively and negatively regulate CD8 T cell infiltration of the brain in discrete neurologic disease models.
TLR9 Knockout in Non-Hematopoietic Cells Protects Mice From Influenza/MRSA Super-infection
Authors: Rich Helen Eva Adams , Martínez-Colón Giovanny , Warheit-Niemi Helen , Gurczynski Stephen , Moore Bethany B
Published: February 2023

Millions of people worldwide are infected with influenza each year, with tens of thousands of deaths yearly in the United States alone. A significant contributor to mortality from influenza is bacterial super-infection from nasal colonizers including S. aureus. Paradoxically, mice lacking the bacterial DNA sensor TLR9 have reduced morbidity and mortality from MRSA super-infection during influenza, concurrent with reduced proinflammatory cytokines in BAL. Surprisingly, macrophages from influenza-infected TLR9−/− mice display increased MRSA phagocytosis and killing compared to macrophages from WT mice. However, bone marrow chimeras reveal that loss of TLR9 on structural (non-hematopoietic) cells is sufficient to reduce bacterial burden to the level of whole-body TLR9−/− mice, while loss of TLR9 in hematopoietic cells does not reduce bacterial burden. Thus, we hypothesize that reduced bacterial recognition by structural cells due to lack of TLR9, mediated by a lower proinflammatory cytokine response, protects animals from influenza/MRSA super-infection. Interestingly, TLR9 may also impact the immune response to influenza through recognizing host mitochondrial DNA. Our data show that influenza induces tlr9 in a wide range of structural and immune cells. Others have shown that influenza induces ER stress, and that influenza infection causes the release of mtDNA into the cytoplasm of lung structural cells. Moreover, others have shown that mtDNA upregulates tlr9 in human lung epithelium. Together, these data suggest that influenza increases mtDNA release inside lung structural cells, which stimulates tlr9 expression in intact lung epithelium, priming the lung for an overexuberant response to bacterial infection.
Pulmonary infection interrupts acute cutaneous wound healing through disruption of chemokine signals
Authors: Crane Meredith , Xu Yun , Monaghan Sean F. , Hall Benjamin M. , Albina Jorge E. , Henry William L. , Tran Holly , Jordon Alexander R.D , Chhabria Karisma , Carlsen Lindsey , Jamieson Amanda M
Published: February 2023

Individuals who are hospitalized with traumatic injuries are susceptible to nosocomial pneumonia. While the effect of the injury on pulmonary infection has been examined in many contexts, it is less well understood how lung infection affects healing wounds. To address this, we examined the outcomes of laparotomized patients with and without pneumonia using the American College of Surgeons NSQIP database. Individuals who developed pneumonia were twice as likely to experience wound dehiscence compared to those without pneumonia. We hypothesized that the immune response could not to meet the demands of competing inflammatory insults in the skin and lungs. To examine this, we developed murine models of post-operative lung infection. Mice were wounded by the dorsal subcutaneous implantation of polyvinyl alcohol (PVA) sponges or tail skin excision, then infected intranasally with Klebsiella oxytoca. PVA sponge wounds allow for the examination of cellular and cytokine responses during acute wound healing, while wound closure can be measured with the tail skin excision model. Pulmonary infection led to rapid suppression of wound IL-1α, IL-1β, and chemokines. This was followed by decreased accumulation of innate leukocytes in the wound and delayed wound closure. In contrast, lung antibacterial responses were not affected by the presence of surgical wounds. Wound healing in infected mice could be rescued by the addition of the chemokines CCL2 and CXCL1; however, this treatment caused a delay in bacterial clearance in the lungs. These data suggest that the immune response is not fully equipped to respond to disparate and competing inflammatory insults, which could have implications beyond the setting of post-operative pneumonia.
Mincle activation and type-1 proinflammatory response during scrub typhus: new insight into innate immune recognition of Orientia tsutsugamushi
Authors: Fisher James Robert , Card Galen , Liang Yuejin , Trent Brandon , Soong Lynn
Published: February 2023

O. tsutsugamushi is an obligate intracellular bacterium and etiologic agent of scrub typhus. The lungs harbor the greatest burden of infection, displaying overzealous, type 1-skewed proinflammatory responses. Here, we investigated whether the C-type lectin receptor (CLR) Mincle contributes to immune recognition and dysregulation. Following lethal infection in C57BL/6 mice, we analyzed pulmonary differential gene expression via the NanoString technology. Of 671 genes analyzed, we found 312 significantly induced genes (adj. p < 0.05) at the terminal phase of disease, among which Mincle (Clec4e) was the 4th greatest up-regulated gene (36 fold compared with mock). In addition, Mincle signaling partners (Fcgrs), Cxcr3, Ccr5, and their ligands, as well as Il27, were all highly up-regulated. To validate a role of Mincle in immune recognition, we exposed murine bone marrow-derived macrophages (MΦ) to live or heat-killed O. tsutsugamushi and analyzed a panel of CLRs and proinflammatory markers via qRT-PCR. While heat-killed bacteria transiently stimulated Mincle transcription that dissipated by 24 hpi, only live bacteria generated a sustained response; infection had limited impact on other tested CLRs. Mincle activation was validated by indirect immunofluorescence and western blot. Live, but not heat-killed, bacteria also generated sustained proinflammatory gene expression in MΦ (Cxcl9, Ccl2, Ccl5, Nos2, Il27). Our study provides the first evidence for selective activation of Mincle in sensing O. tsutsugamushi and a role of Mincle- and IL-27-related pathways in severe infection. Since there are no effective vaccines for scrub typhus, our study helps understand innate immune recognition of this poorly-studied bacterium.
Proliferation of Ly6C+ monocytes/macrophages contributes to their accumulation and inflammation in mouse skin wound healing
Authors: Pang Jingbo , Koh Timothy J.
Published: February 2023

Monocytes and macrophages (Mo/Mφ) play critical roles in all phases of normal skin wound healing. However, persistent accumulation of pro-inflammatory Mo/Mφ contributes to impaired healing in diabetes. The purpose of this study was to determine whether proliferation contributes to Mo/Mφ accumulation in skin wounds. First, proliferating Mo/Mφ (F4/80+Ki67+) were observed in wound cryosections from non-diabetic mice. Next, cell cycle analysis indicated skin wounding increased the number and percentage of Ly6C+F4/80lo/-Mo/Mφ in the S/G2/M phases peaking on day 6 post-wounding with significantly more proliferating cells in wounds from diabetic mice compared to non-diabetic mice. Moreover, unbiased scRNAseq analysis revealed that a cluster dominantly composed of wound Mo/Mφ from diabetic mice exhibited upregulation of genes associated with progression and regulation of cell cycle. Finally, a screen of potential regulators of wound Mo/Mφ proliferation showed increased wound CCL2 levels in diabetic versus non-diabetic mice and that local treatment with CCL2 protein increased proliferation of wound Ly6C+F4/80lo/-Mo/Mφ in non-diabetic mice. Importantly, adoptive transfer of mixtures of CCR2−/− and CCR2+/+ Ly6Chi Mo suggested a critical role for CCL2/CCR2 signaling in promoting Mo/Mφ proliferation in the wound environment. Together, these data demonstrate 1) Ly6C+/F4/80lo/-Mo/Mφ proliferate during skin wound healing with higher rates in wounds from diabetic mice than non-diabetic mice and, 2) a novel role for the CCL2/CCR2 signaling pathway in promoting skin Mo/Mφ proliferation, contributing to persistent accumulation of Mo/Mφ and impaired healing in diabetic mice.
Macrophages activate a MLK3/MEK signaling axis to trigger Annexin mediated membrane repair after cholesterol-dependent cytolysin damage
Authors: Ray Sucharit , Keyel Peter A
Published: February 2023

The innate immune system is critical to promoting host survival during bacterial necrotizing soft tissue infections (NSTIs). NSTI causing pathogens like Streptococcus pyogenes and Clostridium perfringens use pore-forming toxins to kill innate immune cells like macrophages. Toxins cause rapid influx of Ca2+, which triggers repair responses. Ca2+ sensitive proteins like Annexins repair via clogging the injured site and are shed with toxin pores on microvesicles during repair. However, the molecular mechanism orchestrating this repair is unknown. Since Mitogen Activated Protein Kinase (MAPK) pathways initiate long-term inflammatory responses, short-term signaling could drive membrane repair. We hypothesized that Mixed Lineage Kinase 3 (MLK3) activates a prosurvival kinase cascade (MEK/ERK) to activate membrane repair by microvesicle shedding in macrophages. We tested cell viability and repair responses to toxins after blocking proteins in the MAPK pathways using inhibitors or RNAi in primary murine macrophages and cell lines. We found MLK3 or MEK inhibition significantly reduced toxin resistance. Protective MLK3/MEK activity was downstream of Ca2+ and accounted for ~ 60% of all Ca2+ dependent repair. MEK inhibition led to increased cytosolic Ca2+ flux after toxin damage and reduced ANXA2, but not ANXA1 or ANXA6, translocation to damaged site. MEK blockade reduced shedding of toxin pores. Overall, these findings suggest that a Ca2+ dependent MLK3/MEK axis protects macrophages and other cells from pore-forming toxins by recruiting ANXA2 to the cell membrane to promote repair. Modulation of this axis could promote resistance to NSTIs.
Genetically Determined Strength of Natural Killer Cells is Enhanced by Adaptive Admixture of HLA class I Allotypes in East Asians
Authors: Harrison Genelle F , Deng Zhihui , Zhen Jianxin , Zhang Guobin , Chen Rui , Sun Ge , Yu Qiong , Nemat-Gorgani Neda , Guethlein Lisbeth Ann , He Liumei , Tang Mingzhong , Gao Xiaojiang , Cai Siqi , Palmer William , Shortt Jonathan , Gignoux Chris , Carrington Mary , Zhou Hongyan , Parham Peter , Hong Wenxu , Norman Paul
Published: February 2023

Human natural killer (NK) cells are essential for controlling infection, cancer and fetal development. NK cell functions are modulated by interactions between polymorphic inhibitory killer cell immunoglobulin-like receptors (KIR) and polymorphic HLA-A, -B and -C ligands expressed on tissue cells. All HLA-C alleles encode a KIR ligand and contribute to reproduction and immunity. In contrast, only some HLA-A and -B alleles encode KIR ligands and they focus on immunity. By high-resolution analysis of KIR and HLA-A, -B and -C genes, we show that the Chinese Southern Han are significantly enriched for interactions between inhibitory KIR and HLA-A and -B. This enrichment has had substantial input through population admixture with neighboring populations, who contributed HLA class I haplotypes expressing the KIR ligands B*46:01 and B*58:01, which subsequently rose to high frequency by natural selection. Consequently, over 80% of Southern Han HLA haplotypes encode more than one KIR ligand. Complementing the high number of KIR ligands, the Chinese Southern Han KIR locus combines a high frequency of genes expressing potent inhibitory KIR, with a low frequency of those expressing activating KIR. The Southern Han centromeric KIR region encodes strong, conserved, inhibitory HLA-C specific receptors, and the telomeric region provides a high number and diversity of inhibitory HLA-A and -B specific receptors. In all these characteristics, the Southern Han represent other East Asians, whose NK cell repertoires are thus enhanced in quantity, diversity and effector strength, likely through natural selection for resistance to endemic viral infections.
SARS-CoV-2 mediated monocytes dysregulation may contribute to increase risk of cardiovascular risk in severe COVID-19 patients.
Authors: Gunasena Manuja Gayashan Bandara , Wijewantha Yasasvi , Bowman Emily , Gabriel Janelle , Kumar Amrendra , Kettelhut Aaren , Ruwanpathirana Anushka , Weragalaarachchi Krishanthi , Kasturiratna Dhanuja , Vilgelm Anna , Bednash Joseph , Pannu Sonal , Demberg Thorsten , Funderburg Nicholas , Liyanage Namal
Published: February 2023

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS□CoV□ 2) is a highly pathogenic corona virus which causes COVID-19 and resulted in millions of deaths and led to a global public health emergency. An effective and appropriate immune response is essential to control and eliminate viral infections, however, dysregulated immune responses may lead to immunopathology in viral infections. Clinical data showed that COVID-19 may promote the development of cardiovascular disorders (CVDs). However, exact mechanism associated with CVDs in COVID-19 patients is currently unknown. Monocytes are one of the major immune cell subsets that contribute to host defense against many infections but also associated with immunopathology and development of CVDs. Thus, in this study we investigated the role of monocyte subsets in COVID-19 associated CVDs. By using high dimensional flowcytometry, immune biomarker assays and multi variant data analysis in healthy donors (n=17), severe-COVID-19 patients (n=20) (ICU) and COVID-19 recovered individuals (n=30), we found elevated intermediate monocytes and Monocyte chemoattractant protein-1, one of the most important chemokines that regulates migration and infiltration of monocytes to the subendothelial space, where they may become foam cells. Spearman correlation network analysis showed that strong correlation between increase intermediate monocytes in the whole blood and plasma cardimetabolic biomarkers such as fatty acid binding protein 4, C-reactive protein, soluble CD14 and LPS binding protein in ICU patients. Our data show for the first time that the possible role of monocytes in the development of CVDs in ICU patients.
Perforin-2 deficiency delays wound reepithelialization
Authors: O’Neill Katelyn Elizabeth , Padula Laura , Fisher Eva , Head Cheyanne , Wikramanayake Tongyu , Pastar Irena , Tomic-Canic Marjana , Strbo Natasa
Published: February 2023

Wound repair declines with age and contributes to a variety of health complications; however, the cellular and molecular mechanisms governing this decline are not well understood. We observed that the novel pore-forming antimicrobial protein Perforin-2 (P-2) is suppressed in the epidermis of diabetic foot ulcers (DFUs), a major diabetes complication that affects the elderly. Therefore, we sought to investigate the role of P-2 in inducing reepithelialization in wounded skin, particularly focusing on contributions of aging.

We induced wounds on the dorsal skin of 2 or 10-month-old C57/BL6 WT and P-2 deficient (−/−) mice using an 8 mm biopsy punch and assessed healing at day 3 and 6 post wounding. We analyzed reepithelialization and keratinocyte activation using H & E stained and Keratin-6 and Caveolin-1 antibody stained wound tissues, respectively.

We found that wound reepithelialization was delayed in 2-month old P-2 −/− mice on day 3 after wounding in comparison to WT, but there was no difference in reepithelialization by day 6. Interestingly, in 10-month old mice, wound reepithelialization was significantly delayed at day 6 in P-2 −/− mice compared to WT. This delay was accompanied by significantly decreased Keratin-6 expression at the wound edge. Immunofluorescence staining and flow cytometric analysis revealed significantly fewer GD T cells in the wound bed in aged P-2 −/− mice. These P2 −/− GD T cells also displayed significantly lower expression of activation markers such as NKG2D.

Our data demonstrate impaired keratinocyte activation and GD T cell infiltration in the absence of P-2 that is more pronounced in aged mice, indicating a potential role for this protein in inducing the inflammatory response upon wounding.
p38 MAPK activation by Toxoplasma gondii secretory effector protein GRA24: Role in cytokine regulation and induction of protective immunity
Authors: Mercer Heather Lynn , Snyder Lindsay M. , Menard Kayla L. , Doherty Claire M. , Bzik David J. , Fox Barbara A. , Denkers Eric Y.
Published: February 2023

The apicomplexan Toxoplasma gondii induces strong protective immunity in part dependent upon recognition by Toll-like receptors (TLR) 11 and 12 operating in conjunction with MyD88 in the murine host. However, these TLR are not present in humans, prompting us to investigate MyD88-independent signaling pathways of resistance. We show here that parasite dense granule protein GRA24 induces p38 MAPK activation and subsequent IL-12 production in bone marrow derived mouse macrophages (BMDM). Furthermore, Toxoplasma triggers two waves of p38 MAPK phosphorylation in MyD88+/+ and MyD88−/− BMDM. Using the uracil auxotrophic Type I T. gondii strain cps1-1, we demonstrate that the late response does not require active parasite proliferation, but strictly depends upon GRA24. By i. p. inoculation with cps1-1 and cps1-1:Δgra24, we identified unique subsets of chemokines and cytokines that were up and down regulated by GRA24. We demonstrate that cps1-1 triggers a strong host-protective GRA24-dependent Th1 response in the absence of MyD88. To gain deeper insight into genes regulated by GRA24, we employed qPCR to evaluate expression of transcripts for 84 cytokine and chemokine genes in BMDM infected with GRA24-positive compared to GRA24-negative parasites. Our results identify a specific signature of genes regulated by this dense granule protein. In conjunction, using qPCR we evaluated the impact of GRA24 on a panel of macrophage long noncoding RNA (lncRNA) regulated by Toxoplasma infection. Taken together, our data identify GRA24 as a major mediator of p38 MAPK activation, induction of IL-12 and other immune mediators, as well as protective immunity that operates independently of the TLR/MyD88 cascade.
Development of a quantitative COVID-19 Multiplex Assay and its use for serological surveillance in a low SARS-CoV-2 incidence community
Authors: Guarino Cassandra M , Larson Elisabeth , Babasyan Susanna , Rollins Alicia , Joshi Lok , Laverack Melissa , Parrilla Lara , Plocharczyk Elizabeth , Diel Diego , Wagner Bettina
Published: February 2023

A serological COVID-19 Multiplex Assay was developed and validated using serum sample sets from convalescent patients and those collected prior to the onset of the 2020 pandemic. After initial testing of multiple potential antigens expressed in a mammalian system, the SARS-CoV-2 nucleoprotein (NP) and receptor-binding domain (RBD) of the spike protein were selected for the final human COVID-19 Multiplex Assay. A comparison of synthesized and mammalian expressed RBD proteins revealed clear advantages of the mammalian protein. Antibodies directed against NP strongly correlated with SARS-CoV-2 virus neutralization assay titers (rsp = 0.726), while the anti-RBD correlation was moderate (rsp = 0.436). Pan-Ig, IgG, IgA, and IgM against the NP and RBD antigens were evaluated on the validation sample sets. Detection of NP and RBD specific IgG and IgA had outstanding performance (AUC>0.90) for distinguishing patients from controls, but the dynamic range of the IgG assay was substantially greater than for IgA. The COVID-19 Multiplex Assay was then utilized to identify seroprevalence to SARS-CoV-2 in people living in a low-incidence community in Ithaca NY. Samples were taken from a cohort of healthy volunteers (n=332) in early June 2020. Only two volunteers had a positive result on a COVID-19 PCR test performed prior to serum sampling. Serological testing revealed an exposure rate of at least 1.2% (NP) or as high as 5.1% (RBD), which was higher than the measured incidence rate of 0.16% in the county at that time. This highly sensitive and quantitative assay will be useful for continuing to monitor community exposure rates, duration of immune response following infection, and monitoring vaccination responses as vaccines become widely available.
IL-33 activates ILC2 expansion and modulates endometriosis
Authors: Miller Jessica E , Lingegowda Harshavardhan , Symons Lindsey K , Bougie Olga , Young Steven L , Lessey Bruce A , Koti Madhuri , Tayade Chandrakant
Published: February 2023

Introduction: Chronic inflammation drives endometriosis (EM) and its associated symptoms. Interleukin (IL)-33, has gained therapeutic interest as it is elevated in the plasma, peritoneal fluid (PF), and lesions of EM patients. However, how IL-33 contributes to EM is largely unknown. For the first time, we demonstrate, using both human samples and murine models, that IL-33 expands local group 2 innate lymphoid cells (ILC2s) and that IL-33 activated ILC2s modulate hallmark features of EM.

Methods:

We identified ILC2s (CD45+Lin−CRTH2+CD127+ST2+) in the PF of EM patients using flow cytometry. Using immunofluorescence, we determine whether IL-33 forms epithelial or stromal cell niches within the EM lesion. Then, using our well established EM model, we induced EM in female C57Bl/6 mice and treated with PBS or IL-33. Hallmark features of EM were assessed including inflammation (using multiplex cytokine analysis), immune cell profiling (using flow cytometry), and lesion architecture (using immunohistochemistry and Nanostring transcriptomic profiling). To establish cause and effect, we treated ILC2 deficient mice with PBS (n=5) or IL-33 (n=5). Unpaired t-tests and one-way ANOVAs were done when appropriate.

Results/Discussion:

In humans, ILC2s are present and elevated in EM patients (n=5) compared to healthy donors (n=3) and IL-33 colocalized with both epithelial and stromal cells within the EM lesion. Using murine models, IL-33 drove hallmark features of EM including increased inflammation, alterations in PF immune cells (including eosinophils, T cells, macrophages and ILC2s), as well as increased lesion proliferation, neurogenesis and fibrosis. Using ILC2 deficient mice, we show that IL-33 induced pathology is ILC2 dependent.
Adipocyte-specific PIK3C3/VPS34 controls adipogenesis, metabolically-triggered inflammation, insulin resistance, and adaptive thermogenesis in mice
Authors: Song Wenqiang , Yang Guan , Postoak J. Luke , Zhang Jianhua , Wu Lan , Van Kaer Luc
Published: February 2023

Interactions between cells of the immune and metabolic systems play critical roles in controlling immune responsiveness and metabolic health. When these interactions are dysregulated, metabolic diseases such as obesity or lipodystrophy may develop. We have focused on cellular processes related to autophagy, a cellular self-degradation process, in controlling interactions between the immune and metabolic systems. In this study, we analyzed mice with an adipocyte-selective deficiency in PIK3C3/VPS34, a key early player in autophagy. These animals showed reduced white and brown adipose tissue (WAT and BAT) with enhanced macrophage infiltration, systemic immune activation, and spontaneous insulin-resistance. The phenotype of these animals bears a striking resemblance with the disease manifestations seen in individuals with lipodystrophy. Mechanistically, we found that conditional Pik3c3-deficiency inhibits adipogenesis in both WAT and BAT by downregulating glucose uptake and fatty acid metabolism, and impairs the thermogenic function of BAT by downregulating the expression of key regulators (Prdm16, Ucp1, and Cidea). Upon acute cold exposure, Pik3c3-deficient mice became cold-sensitive under fasting conditions due to insufficient thermogenesis in BAT. In contrast, these animals exhibited blunted beige fat thermogenesis in subcutaneous WAT in response to administration of a b3-adrenergic receptor agonist. Collectively, our data demonstrate that PIK3C3 is a vital regulator of adipose tissue development, metabolically-triggered inflammation, insulin resistance, and adaptive thermogenesis. Hence, we identify PIK3C3 as a potential therapeutic target for metabolic diseases such as type 2 diabetes.
Meningeal lymphatics near the cribriform plate undergo lymphangiogenesis and directly engage in leukocyte crosstalk during neuroinflammation
Authors: Hsu Martin , Madrid Andy , Laaker Collin , Choi Yun Hwa , Herbath Melinda , Sandor Matyas , Fabry Zsuzsanna
Published: February 2023

Meningeal lymphatics in the dural layer surrounding the dorsal regions of the brain, basal regions, and near the cribriform plate have all been implicated in the management of neuroinflammation and edema by facilitating the drainage of fluid, macromolecules, antigens, and cells. We have previously published that during neuroinflammation only the lymphatics near the cribriform plate are able to undergo extensive morphological changes through lymphangiogenesis in response to neuroinflammation during Experimental Autoimmune Encephalomyelitis (EAE), a mouse model of Multiple Sclerosis. In this study, we hypothesize that these cribriform plate lymphatics likely have other phenotypic changes as a consequence of neuroinflammation-induced lymphangiogenesis, and that identifying these phenotypic alterations may shed light on potentially novel roles of meningeal lymphatics during neuroinflammation. Single cell RNA sequencing reveals the upregulation of genes involved in leukocyte crosstalk and regulation such as chemotaxis/adhesion and antigen processing/presentation, suggesting that cribriform plate lymphatics may play a direct role in regulating adaptive immunity in addition to its canonical roles in facilitating drainage. We also demonstrate that cribriform plate lymphatics are able to capture CNS-derived antigens and functionally engage in crosstalk with dendritic cells and CD4 T cells. These data characterize cribriform plate lymphatics and demonstrate that these vessels become dynamic in response to neuroinflammation to facilitate excess drainage as well as directly modulate immunity through leukocyte crosstalk.
Sodium chloride generates anti-inflammatory Th17 cell responses but amplifies Th17 cell pathogenicity in pro-inflammatory cytokine microenvironments
Authors: Zielinski Christina , Matthias Julia
Published: February 2023

Ionic signals have recently been demonstrated to affect T cell polarization and function. Sodium chloride (NaCl) was proposed to accumulate in peripheral tissues upon dietary intake and to promote autoimmunity via the Th17 cell axis. We here demonstrate that together with amplification of human Th17 cell signature properties, NaCl gave rise to a stable anti-inflammatory Th17 cell fate that we found to be restricted to distinct pathogen specificities. The p38/MAPK pathway involving NFAT5 and SGK1regulated FoxP3 and IL-17 expression in high NaCl conditions. The NaCl induced acquisition of an anti-inflammatory Th17 cell fate was confirmed in vivo in the mouse model of experimental autoimmune encephalomyelitis (EAE), which demonstrated strongly reduced disease symptoms upon transfer of NaCl-treated encephalitogenic Th17 cells. However, NaCl was coopted to promote murine and human Th17 cell pathogenicity, if T cell stimulation occurred in a proinflammatory and TGF-b low cytokine microenvironment. This revealed a context dependent dichotomous role for NaCl in shaping the pathogenicity of Th17 cells. NaCl might therefore prove beneficial for the treatment of chronic inflammatory diseases in collaboration with cytokine blocking drugs.
Regulation of TLR9 signaling by N-glycosylation of UNC93B1
Authors: Song Hyun-Sup , Huh Ji-Won , Lee Yu-Ran , Jung Da-Jung , Yang Chorong , Kim You-Me
Published: February 2023

Nucleotide-sensing Toll-like receptors (TLRs), such as TLR3, 7, and 9, reside in the endolysosomal compartments to avoid activation by host DNA or RNA. Proper intracellular localization and signaling by these TLRs depend on the physical interaction with UNC93B1. Specific amino acid residues in UNC93B1 have been identified to affect the functions of endosolysomal TLRs. However, it is largely unknown how UNC93B1 differentially regulates individual TLRs.

Protein N-glycosylation controls folding, maturation, stability, localization and protein-interaction of target glycoproteins. In this study, we examined whether the N-glycosylation of UNC93B1 affects its properties and molecular function.

Using mutagenesis, we found that the N251 and N272 residues of UNC93B1 become N-glycosylated. We expressed wild type (WT) and N-glycosylation-defective UNC93B1 mutants (N251Q and N272Q) in UNC93B1-deficient cells and found that signaling of TLR9, but not that of other TLRs, were defective in UNC93B1 N272Q-expressing cells. The N272Q mutation did not affect the protein stability, localization, interaction of UNC93B1 and TLRs, nor did it prevent the CpG DNA-binding of TLR9. Nonetheless, upon CpG DNA stimulation, the recruitment of MyD88 to TLR9 was significantly inhibited in UNC93B1 N272Q-expressing cells compared to WT cells. Consequently, poor phosphorylation and degradation of I_B were observed in UNC93B1 N272Q-expressing cells. Combined, our data show that N-glycosylation of UNC93B1 specifically regulates TLR9 signaling by modulating the recruitment of MyD88 to TLR9.
In silico mapping of 14-3-3ζ and Human TRAF interaction
Authors: Gupta Anish U , Gupta Shrey , McGowan Jenna , Chakravarti Ritu
Published: February 2023

Recent advances show that the 14-3-3ζ protein participates in several immune regulations and is a unique regulator of IL-17A signal transduction. The IL-17A signal transduction triggers a 14-3-3ζ-TRAF5 dependent and a 14-3-3ζ-TRAF6 dependent intracellular pathway responsible for producing CXCL-1 and IL-6, respectively. Improved IL-17A signaling blockers are desirable in treating autoimmune diseases such as rheumatoid arthritis and lupus. Due to its unique role, 14-3-3ζ is an attractive target to regulate CXCL-1 and IL-6 levels. The aim of this study is to determine interaction sites between the 14-3-3ζ and TRAF (5 and 6) proteins using bioinformatic tools. We used 6EF5.pdb for 14-3-3ζ, 4GJH.pdb for TRAF5, and ILB6.pdb for TRAF6. Using ZDock, we examined the putative site of the 14-3-3ζ interactions on TRAF (5 and 6) with restrictions and without restrictions. The mapped interacting residues were mutated, and an effect on the interaction with 14-3-3ζ was observed. To further evaluate the interaction quality, we utilized Prodigy to measure the binding energy for several possible structures and narrow down the selected interaction sites further. Results indicate that site 479–485 is the putative target site of the 14-3-3ζ-TRAF5 complex with a ΔG of −10.1 kcal/mol and a Kd of 4.00×10−8. By comparison, residues 483–488 on TRAF6 interact with 14-3-3ζ with a ΔG of −19.1 kcal/mol and a Kd of 9.50×10−15. Our results demonstrate the putative binding sites between the 14-3-3ζ and TRAF (5 and 6) proteins. However, our study has the limitation of using the available but incomplete TRAF structures; therefore, lab studies should be used to confirm these results. Our results provide a rationale to investigate the 14-3-3ζ and TRAF proteins further.
A role of B-cells and antibodies in generation of protective immune responses against a vaccinating antigen during non-albicans Candida mediated systemic candidiasis
Authors: Rohatgi Soma , Shukla Manisha
Published: February 2023

While cellular immunity is acknowledged to be critical for vaccine-mediated protection during systemic candidiasis, the role of humoral immunity is not clear. We investigated the role of B cells and antibodies in conferring protective advantage during Candida tropicalis mediated murine systemic candidiasis, using a lead vaccine candidate, Sap2 (secreted aspartyl proteinase 2) obtained from C. parapsilosis. Compared to sham-immunized mice, Sap2-parapsilosis vaccinated mice exhibited significantly improved survival outcomes (p=0.02) and reduced organ fungal burdens (p<0.05). Sap2-parapsilosis vaccination induced high titres of Sap2-specific antibodies, and a fraction of antibodies could bind whole fungus (of predominantly IgM isotype). Notably, sera from Sap2-parapsilosis-vaccinated mice exhibited increased C. tropicalis biofilm inhibition ability and enhanced neutrophil-mediated fungal killing in vitro. Sap2-specificity was further confirmed by depleting antigen-specific antibodies. Passive transfer of Sap2-parapsilosis immune serum significantly reduced fungal burdens in naive mice, as compared to mice receiving sham-immune serum, upon infection. Higher numbers of total CD19+ B cells, plasma cells and Candida-binding B cells in Sap2-vaccinated mice indicate towards a role of B cells during early stages of Sap2-mediated immune response. Epitope analysis performed using identified B cell epitopes provides insights about including important IgM and IgG epitopes, when designing multivalent or multiepitope anti-Candida vaccine/s. In summary, our results establishes a role of B cells and antibodies in contributing towards protective efficacy of Sap2 vaccine antigen during murine systemic candidiasis.
Metallothioneins reprograms T cell response towards pulmonary Histoplasma capsulatum through altering their metabolic profile
Authors: Alrefai Hani , Vignesh Kavitha Subramanian , Deepe George S
Published: February 2023

Histoplasma capsulatum (Hc) is a dimorphic fungus that is affected by environmental temperature. Hc is distributed worldwide. When inhaled, the microconidia and spores reach the lung and are converted to the pathogenic yeast phase within the phagosomes of macrophages. The Hc replicates intracellularly until these phagocytes are activated by cytokines, typically from activated T cells. Excluding iron, the effect of trace metals on Hc interaction with innate immune cells has been inadequately studied. The cells’ metabolic processes are altered, and the cytokine macro/microenvironment change in the pathogen’s face. Zn2+ metalloproteins constitute about 10% of the mammalian proteome, and many transcription factors and enzymes use this metal. Metalloproteins are linked to many energy-producing pathways, including carbohydrate metabolism, amino acid metabolism, and the redox chain. Our experiments showed that ablation of Metallothioneins 1 and 2 (MT1/2) decreases the immune system’s ability to clear Hc from the lungs. The numbers of T cells are not affected by the MT1/2 ablation; rather, the cells’ activity is decreased. MT1/2 ablated dendritic cells (DC) are not defective. MT1/2 ablated CD4 T cells show an alternative metabolic profile. MT1/2 ablated CD4 T cells show a decrease in their genome acetylation.
Pseudomonas aeruginosa Outer Membrane Vesicles (OMVs) Cause Inflammation and Cardiomyocyte Contractile Dysfunction
Authors: Rajaram Murugesan , Kumar Naresh , Pestrak Matthew Pestrak , Wu Qian , Nolan Sheri Dellos , Lafuse William , Ganesan Latha P. , Wozniak Daniel J.
Published: February 2023

P. aeruginosa (P.a) infection causes severe pneumonia, which can lead to cardiac inflammation and severe cardiac complication. Growing evidence indicates that cardiac resident macrophages are important regulators of inflammation and steady state heart function. On the other hand, pathogen secreted OMVs carry bacterial immunogens that activate the host immune system and alter the homeostasis of normal organ function. Our published results showed that P.a. infection causes cardiac inflammation, fibrosis, and cardiac dysfunction without the bacteria disseminating into heart tissue. However, the mechanism of P.a. induced cardiac inflammation and dysfunction is not known and understanding this is critical for the development of host directed therapy. Thus, we hypothesize that cardiac damage caused by P.a infection is mainly due to the release of bacterial toxins and/or OMVs or by inflammatory mediators released by immune cells. To test our hypothesis we have used human monocyte derived macrophages (hMDMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Our results show that conditioned media from P.a. infected hMDMs causes contractile dysfunction in hiPSC-CMs. In addition, we found that P.a. OMVs induce similar contractile dysfunction in hiPSC-CMs. Notably, we also found that OMV stimulation activates classical inflammatory signaling pathways and enhances hMDM inflammatory cytokine production. These findings demonstrated that P.a OMVs induce inflammatory cytokine production and subsequent myocyte contractile dysfunction. Studies are ongoing to determine the mechanism of OMV induced contractile dysfunction as well as the impact of OMVs on cardiac dysfunction in a mouse model.
Complement Receptor 3, but not Fcγ Receptor III, abolishes the intracellular survival advantage of virulent Rhodococcus equi in murine macrophages
Authors: Silveira Bibiana Petri da , Cohen Noah D. , Bray Jocelyne M. , Barhoumi Rola , Bordin Angela Ilha
Published: February 2023

Complement receptor 3 (CR3) and Fc gamma receptor III (FcγRIII) are proposed to be essential for mediating Rhodococcus equi entry and killing in macrophages, respectively. Our objective was to investigate the role of each of these receptors in phagocytosis and intracellular replication of R. equi by macrophages. Wild-type (WT), CR3−/−, and FcγRIII−/− J774A.1 macrophages were infected with either virulent or avirulent opsonized R. equi. Quantitative culture was performed immediately post-infection (T0) and after 48 h (T48). Data were analyzed using mixed-effects modeling with the following outcomes: 1) ratio of inoculum:T0 (phagocytosis); and, 2) ratio of T48:T0 (intracellular survival). Additionally, WT, CR3−/−, and FcγRIII−/− macrophages were infected with virulent or avirulent GFP+ R. equi to confirm intracellular infection by confocal microscopy. Our results demonstrate that phagocytosis of either virulent or avirulent R. equi was unaffected by knocking out CR3 or FcγRIII, and imaging revealed that CR3−/− and FcγRIII−/− internalized both R. equi strains similarly to WT macrophages. As expected, intracellular survival in WT was significantly higher for virulent than avirulent R. equi. In CR3−/−, however, no significant difference in survival between virulent and avirulent strains was observed. Knocking out FcγRIII did not alter R. equi intracellular replication. Our findings demonstrate that CR3 is not necessary for R. equi infection and show that CR3 deletion eliminated the intracellular survival advantage of virulent strains. Last, FcγRIII-mediated phagocytosis does not improve intracellular killing as previously suggested.