bluuebunny/crossref_metadata_2025 huggingface dataset:
IRF4 regulates the proliferative response during B cell differentiation in vivo
Authors: Patterson Dillon , Scharer Christopher , Mi Tian , Hicks Sakeenah L , Zhang Qiang , Boss Jeremy M
Published: January 2023

Cell division is an essential component of B cell differentiation to a plasma cell (PC). Division coupled changes in the expression of transcription factors that coordinate the PC program are known; however, little is known regarding the timing/extent of reprogramming by these factors as the cells are dividing in vivo. Furthermore, how/when these factors coordinate cell division during differentiation has remained unexplored. To address this, we assessed the cell division kinetics of wild-type (WT) B cells responding to LPS in vivo. Interestingly, we found that WT B cells must undergo 8 divisions before differentiating into PCs. Computational modeling of division rates defined a proliferative burst between 48 and 60 hours after LPS injection that is characterized by a rapid increase in the rate of cell division. In contrast, Interferon Regulatory Factor 4-deficient (IRF4−/−) B cells divided but stalled at divisions 2–6, failing to undergo the proliferative burst. To assess the timing/scope of IRF4-dependent reprogramming, WT and IRF4−/− responding B cells at divisions 0, 1, and 3–6 were sorted for ATAC- and RNA-seq. RNA-seq data revealed hundreds of differentially expressed genes (DEGs) when IRF4 was deleted, a subset of which included Myc target genes. ATAC-seq data also exposed hundreds of differentially accessible regions (DARs), the majority of which contained a known IRF4 binding motif and mapped to a corresponding DEG. Interestingly, IRF4−/− B cells progressively became more divergent as they divided when compared to WT B cells in the same division. Together, these data create a road map defining the role of IRF4 throughout B cell differentiation and reveal a critical role for IRF4 in maintaining the proliferative response.
Hypoxia and TGF-β1 induce tissue resident memory phenotype cells from human peripheral blood T-cells in vitro
Authors: Hasan Farah , Shaw Rebecca , Wang Junmei , Yee Cassian
Published: January 2023

Tissue resident memory cells (TRM) are a recently identified subset of memory T-cells important in frontline defense against viral diseases and recent reports suggest T-cells with this phenotype are important in anti-tumor immunity. However, relatively little is known regarding TRM differentiation and endogenous TRM are notoriously difficult to isolate, impeding their study in basic research and downstream applications. Thus a means to generate TRM in vitro would be desirable. It is known that TGF-β is critical in establishing TRM populations and attempts to identify other factors that can induce differentiation to resident phenotype have focused on cytokines. Oxygen tension is another factor that distinguishes the circulation from peripheral tissues; thus we hypothesized that hypoxia may contribute to a TGF-β1-induced TRM phenotype in human CD8+ T-cells. We demonstrate that differentiation of human peripheral blood CD8+ T-cells in hypoxia and TGF-β1 in vitro led to the development of a TRM phenotype, characterized by a significant increase in CD69+CD103+ cells expressing CD49a, CD101, and PD-1 – hallmarks of human TRM. At the transcriptional level we observed upregulation of genes associated with TRM differentiation and downregulation of genes associated with memory cell recirculation. Hypoxia and TGF-β1 appear to synergize to induce a resident phenotype as their combination produced a significantly larger population of TRM phenotype cells than the additive effects of either condition alone. Our findings identify a previously unreported cue for TRM differentiation and enable a facile means of generating human TRM phenotype cells in vitro for basic studies and translational applications such as adoptive cellular therapies.
Identity of recirculating T cells i humans
Authors: Buggert Marcus , Nguyen Son , Darko Samuel , Ransier Amy , Douek Daniel , Björkström Niklas K. , Dori Yoav , Itkin Max G. , Betts Michael R
Published: January 2023

Memory T cells are widely considered to continually recirculate between tissues and blood via lymph to maintain immunosurveillance. While much effort has been dedicated to understand which memory T cells that are present in tissues, less is known about peripheral blood memory T cell subsets that recirculate through and egress from tissues during steady-state. Here we directly identify the tissue egressing (recirculating) immune system by sampling thoracic duct lymph (TDL), blood and peripheral tissues in humans and primates. We find that 70% of all immune cells egressing from tissues are T cells. Through transcriptional, functional, clonotypic, and epigenetic profiling, we find that cytolytic memory CD8+ T cell subsets are almost entirely confined to blood, while their phenotypic counterparts in TDL and many tissues represent non-cytolytic cells with higher regenerative capacity. Cytolytic T cells in blood possess a distinct clonotype distribution compared to TDL CCR7- T cells and are retained in blood after treatment with the tissue egress inhibitor fingolimod. We additionally find that HIV-specific CD8+ T cells can be readily found in TDL but again lack a cytolytic phenotype. We propose that cytolytic molecule expression is confined to a blood-resident memory T cell population in steady-state and that the intermediate differentiation status of HIV-specific CD8+ T cells is a fingerprint of their systemic recirculation capacity, rather than dysfunction, in humans.
Role of the Microbiota in Primary Lung Cancer Initiation and Progression
Authors: Rose uriel D , Vetizou Marie , Roy Soumen , Edwards Bathai , Peck Misty , Smith Loretta , Robles Ana , Harris Curtis , Difilippantonio Simone , Trinchieri Giorgio
Published: January 2023

Lung cancer is the leading cause of cancer-related death in men in 87 countries and in women in 26 countries. The commensal microbiota lives on the epithelial barriers of the host and is vital for the health of the organism. Specific microbial species have been described to affect neoplastic pathology both at the gastrointestinal tract level and systematically in organs that are not normally associated with the gut microbiota. Studies have revealed the importance of the gut microbiota in the efficacy of anti-cancer therapies (chemo and immunotherapy) However, the role of lung microbiome in lung cancer remains largely unknown. We aimed to study the role of the lung microbiota during lung carcinogenesis progression in the context of p53/K-ras mutation and its impact on the tumor microenvironment. To further analyze the impact of the lung microbiota on lung cancer initiation, P53-Kras mutant germ-free mice will be colonized by intranasal instillation with bacteria identified in human lungs samples from lung cancer patients such as Variovorax paradoxus (V.paradoxus) and Acidovorax temperans (A.temperans) and the tumor burden will be calculated. Our data indicates Germ-free mice instilled with V. paradoxus trended towards an increase tumor burden compared to respective controls while A. temperans -associated germ-free mice seems to delay tumor growth. Germ-free mice, at 18 and 24 weeks post-Ad-Cre virus instillation, tends to have an increased tumor burden than their SPF counterparts. Also, changes in immune infiltrate were observed after flow cytometric analysis with preliminary data showing a decrease in CD45 positives cells the cells in mice instilled with V. paradoxus as well as an increase in Monocytes and Macrophages populations.
Induction of a long non-coding RNA, lncRNA-Chr1:1226, by Cryptosporidium infection primes intestinal epithelial cells for IFN-γ-mediated host antimicrobial gene transcription
Authors: Chen Xian-Ming , Deng Silu , Gong Ai-Yu , Wang Yang , Zhang Xin-Tian , Li Min , Li Juan , Mathy Nicholas W
Published: January 2023

Cryptosporidium, a protozoan parasite that infects the intestinal epithelium and other mucosal surfaces in animals and humans, is an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children in developing countries. Intestinal epithelial cellular defense is key to innate mucosal anti-Cryptosporidium defense but underlying molecular mechanisms are still obscure. Here, we identified several long non-coding RNAs (lncRNAs) that are predominantly expressed in intestinal epithelial cells. Several of such epithelial-enriched lncRNAs, such as lncRNA-Chr1:1226, were upregulated in cells following C. parvum infection. Induction of lncRNA-Chr1:1226 in infected intestinal epithelial cells was controlled by TLR4/NF-κB/Cdc42 signaling and epithelial specific transcription factor Eif3. Induction of lncRNA-Chr1:1226 promoted IFN-γ-mediated epithelial antimicrobial defense, through facilitating STAT1/SWI/SNF-associated chromatin remodeling to promote IFN-γ-mediated transcription of defense genes in intestinal epithelial cells. We observed that IFN-γ-mediated antimicrobial defense was suppressed in neonatal intestinal epithelium. Expression of PRDM1 in the neonatal intestinal epithelium might contribute to suppression of IFN-γ-mediated antimicrobial gene transcription. Furthermore, PRDM1 interacted with lncRNA-Chr1:1226 and PIAS1 to attenuate SWI/SNF-mediated antimicrobial transcription induced by IFN-γ in intestinal epithelium of neonates. Our data demonstrate that lncRNAs, particularly epithelial lncRNAs, may be key regulators in IFN-γ-mediated epithelial defense.
Infiltrated monocyte and T cell interaction drives immunopathology in the brain during cryptococcal meningoencephalitis
Authors: Xu Jintao , Ivey Mike , Koble Jessica , Lopez Rafael , Mechler Claire , Neal Lori , Williamson Peter , Segal Benjamin , Olszewski Michal A
Published: January 2023

Cryptococcal meningoencephalitis (CM) is a major cause of central nervous system (CNS)-linked mortality worldwide. High mortality frequently occurs despite antifungal drug treatment and pathogen elimination, and increasing evidence suggests over-exuberant host immune responses promote CM pathogenesis. Our goal was to dissect the role of two major groups of cells recruited to CNS (myeloid cell and T cell) and determine whether and how their crosstalk drives CM immunopathology.

To study this, we developed a murine model that reproduces major features of human CM. Using this model, we found mice mortality did not directly correlate with CNS fungal burden, but instead was synchronized with the accumulation of ultra-Th1 polarized CD4 T cells and CCR2+ monocytes (Mo). Recruitment of Mo into the CNS was impaired in CCR2−/− mice, and these mice survived significantly longer and displayed fewer neurological symptoms despite higher brain fungal burden compared to WT mice. Mechanistically, we found that 1) CCR2+ Mo contributed to CXCR3+IFNγhi CD4 T cell accumulation and their ultra-Th1 polarization through modulating CXCL9/CXCL10 production. 2) CXCR3 deletion resulted in diminished CD4 T cell recruitment, Th1 polarization and conferred markedly protection against CM mortality; 3) robust IFNγ production by CD4 T cells corresponded to massive iNOS expression in Mo, which propose excessive NO production potentially contributes to neuronal injury. Collectively, we showed CCR2+ Mo and CXCR3+ CD4 T cells interact to promote mutual recruitment and activation, causing profound CNS damage during CM. Regulation of Mo and T cell function may become therapeutic strategy to aid current anti-fungal drug treatment that is frequently ineffective.
Microbial Activation of B-cells by Pattern Recognition Receptors Contributes to the Proinflammatory and Profibrotic Milieu in Patients with Idiopathic Pulmonary Fibrosis
Authors: Ali Mohamed , Dasari Harika , Van Keulen Virginia , Egan Ashley , Peikert Tobias , Carmona Eva M
Published: January 2023

We hypothesize that B-lymphocytes are conducive to the pathogenesis of idiopathic pulmonary fibrosis (IPF), a progressive and fatal interstitial lung disease. B-lymphocytes aggregate in the lungs of IPF patients to form tertiary lymphoid follicles. How these follicles are formed and their role in the pathogenesis of IPF is not known. Hence, we sought to characterize how the activation of B-lymphocytes from IPF patients may contribute to the disease.

Methods

IPF patients defined by ATS guidelines were identified and recruited under local IRB #14-004714. B-lymphocytes were then isolated by negative selection. Cytokines and immunoglobulins from plasma or B-lymphocyte supernatants were measured by ELISA and signaling pathways were elucidated from whole-cell lysates by immunoblotting. Our results show that B-lymphocyte aggregates are present within fibrotic areas of the lung of IPF patients and that activated B-lymphocytes induce fibroblast migration. This effect was abrogated by nintedanib inhibition of B-lymphocyte activation. Furthermore, microbial antigen (β-glucan and CpG) activation of B-lymphocytes through pattern recognition receptors (PRRs; TLR9 and Dectin-1) contributed to the proinflammatory and profibrotic milieu seen in IPF patients. Stimulation of TLR9 and Dectin-1 resulted in activation of mTOR-dependent and -independent pathways, respectively. Rapamycin decreased TLR9 signaling, but not Dectin-1 mediated pathways. Nintedanib abrogated B-cell activation, IgM production, and the profibrotic milieu by interfering with both the mTOR-dependent and -independent pathways. In conclusion, our findings implicate activated B-lymphocytes in IPF pathogenesis highlighting the role of PRRs in this disease.
Long Pentraxin 3 (PTX3) Regulates IL-17A Mediated Immunity to Primary and Secondary Leishmania major Infection
Authors: Gupta Gaurav , Jia Ping , Sharma Rohit , Zayats Romaniya , Shan Lianyu , Mou Zhirong , Murooka Thomas , Soussi-Gounni Abdelilah , de Oliveira Camila I , Uzonna Jude E
Published: January 2023

Resolution of cutaneous leishmaniasis (CL), caused by the protozoan parasite Leishmania (L) major, relies on the nature of the host immune response. The long pentraxin 3 (PTX3), a soluble pattern recognition molecule, is critical for wound healing by regulating tissue repair and also in innate and adaptive responses during infection and inflammation. Here, we show that PTX3 regulates disease pathogenesis and immunity to CL. PTX3 expression is increased in active skin lesions in patients and mice during CL, with higher levels being expressed in individuals with severe disease. PTX3−/− mice were highly resistant to primary and secondary L. major infection. Interestingly, the enhanced resistance of PTX3−/− mice to primary or secondary L. major infection was not associated with enhanced IFN-γ or decreased IL-10 response. Instead, L. major-infected PTX3−/− mice displayed strong IL-17 response and in vivo neutralization of IL-17A abolished their enhanced resistance. Naïve CD4+ T cells from PTX3−/− mice displayed increased differentiation into Th17 cells and this was associated with increased expression of Th17-specific transcription factors including RORγt, AhR and STAT3. Addition of recombinant PTX3 significantly inhibited the expression of Th17-specific transcription factors and the frequency of Th17 cells in Th17 polarizing cultures of PTX3−/− CD4+ T cells. Similarly, healed PTX3−/− mice had higher frequencies of effector memory CD4+ T cells that rapidly proliferate and differentiate into IL-17A-producing CD4+ T cells upon secondary challenge. Collectively, our results show that PTX3 contributes to pathogenesis of CL by negatively regulating protective Th17 and IL-17 responses.
Gamma Delta and Natural Killer cells in Splenocytes of Young mice Protect Immunocompromised mice from Death in Plasmodium chabaudi Infection
Authors: Richard Lyndsay B , Smith Margaret R. , Murrray Annie E , Opata Michael M
Published: January 2023

Immunity to malaria requires an elongated time to develop and understanding of the immune response to malaria, especially in children under five, is limited due to the lack of a reliable animal model to study the pathogenesis of the disease. By utilizing a newly developed young rodent model in our lab, we have observed that purified CD4+ T cells from 8-week adult mice proliferate faster than day 15 old young mice (pups) when stimulated in vitro. Surprisingly, we observed faster proliferation of splenocytes in the pups, when compared to the cells from adult mice. When transferred to immunocompromised RAGKO mice, both pup and adult cells protected from death. These data suggest that apart from CD4+ T cells, there is a faster responsive cell population in the pups that may promote protection against malaria infection in mice. To better understand this responsive cell population in young mice, we adoptively transferred splenocytes from both adults and pups into immunocompromised RAGKO mice and infected these cell recipients with P. chabaudi. We observed higher proportions and numbers of gamma delta (gd) T cells in the pup splenocyte recipients when compared to adult counterparts on day 8 post-infection. Interestingly, we observed lower proportions and numbers of natural killer (NK) cells in the adult cell recipients compared to pup recipients. However, there were significantly higher proportions, but not numbers of macrophages in the adult cell recipients. Taken together, our findings suggest that pup cells are enriched or promote an innate immune response comprising of gd and NK cells, while adult cells inhibit the expansion of these innate lymphocyte cells in malaria infection.
Pulmonary inflammation and Tie2/Angiopoietin mediated vascular dysregulation during lethal Orientia tsutsugamushi infection
Authors: Trent Brandon J , Liang Yuejin , Xing Yan , Esqueda Marisol , Yang Wei , Cho Nam-Hyuk , Kim Hong-Il , Kim Yeon-Sook , Shelite Thomas R , Cai Jiyang , Sung Jiaren , Bouyer Donald , Liu Peter , Soong Lynn
Published: January 2023

Orientia tsutsugamushi infection can cause acute lung injury and high mortality in humans; however, the underlying mechanisms are unclear. Here, we tested a hypothesis that dysregulated pulmonary inflammation and Tie2-mediated endothelial malfunction contribute to lung damage. Using a murine model of lethal O. tsutsugamushi infection, we demonstrated pathological characteristics of lung damage: 1) a significant increase of ICAM-1, VEGFR2, and angiopoietin-2 (Ang2) proteins in inflamed tissues and lung-derived endothelial cells (EC), 2) a progressive loss of endothelial quiescent and junction proteins (Ang1, VE-cadherin), and 3) a profound impairment of Tie2 receptor at the transcriptional and functional levels. In vitro infection of primary human EC cultures and serum Ang2 proteins in scrub typhus patients support our animal studies, implying endothelial dysfunction in severe scrub typhus. Evidence for neutrophil-mediated inflammation and tissue damage included in vitro infection and activation of neutrophils, lung accumulation of activated (CD63+)neutrophils, co-localization of lung bacteria with neutrophils, and co-localization of MPO+ neutrophils with Ang2+ ECs. At the onset of disease, pulmonary macrophages (MF) were polarized toward an M1-like phenotype, with limited detection of M2-like cells. Comparative studies of LPS- versus IL-4-primed MFs revealed extensive bacterial replication in M2-type MFs that preferentially expressed the IL-10 and SOCS1 genes. This is the first detailed investigation of lung cellular immune responses during acute O. tsutsugamushi infection and highlights inflammatory biomarkers future therapeutic research for the control of this neglected tropical disease.
Pre-existing SIV infection decreases cytokine responses by T cells in lung during the early stages of M. tuberculosis co-infection
Authors: Larson Erica C , Rodgers Mark A , Ellis Amy L , Ameel Cassaundra L , Baranowski Tonilynn M , Balgeman Alexis J , Maiello Pauline A , O’Connor Shelby L , Scanga Charles A
Published: January 2023

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death among HIV+ individuals. The precise mechanisms by which HIV impairs the immune response to subsequent Mtb infection are unknown. We previously established a model of co-infection in Mauritian cynomolgus macaques (MCM). We found SIV/Mtb co-infected MCM had rapidly progressive TB and reached humane endpoint by 12 weeks post-Mtb infection. We hypothesized that pre-existing SIV impacts T cell responses at the early stages of Mtb infection. We infected MCM with SIVmac239 intrarectally and 6 months later co-infected with a low dose of Mtb. SIV-naïve MCM were infected with Mtb alone as controls. Animals were monitored by clinical parameters, culturing bacilli in gastric and bronchoalveolar lavages, and serial 18F-FDG PET/CT imaging. Six weeks after Mtb infection, animals were necropsied and immune responses in the lung were measured by flow cytometry. The two groups exhibited similar TB disease at time of necropsy. Total bacterial burden was remarkably high in both SIV-naïve and SIV/Mtb groups, although co-infected animals tended to have more bacteria in lung tissue. At sites of Mtb infection, SIV/Mtb co-infected animals had fewer CD4+ T cells and significantly more CD8+ T cells. TNFα production by these CD4+ and CD8+ T cells was decreased. Taken together, pre-existing SIV decreases the quality of cytokine responses by T cell at sites of Mtb infection during the early stages of TB disease. This appears to be a critical time point at which the immunologic defect from pre-existing SIV infection begins to impact the arc of TB disease.
Wnt4 controls early cDC1 commitment to suppress Type 2 immunity
Authors: Hung Li-Yin , Johnson John L , Ji Yingbiao , Christian David A , Herbine Karl R , Pastore Christopher F , Herbert De’Broski R
Published: January 2023

Whether conventional dendritic cells (cDC) acquire subset identity under direction of regenerative glycoproteins is unknown. We demonstrate that Wnt4, a beta-catenin independent Wnt ligand, is necessary and sufficient for pre-cDC1 BM specification. CD11c-restricted Wnt4 deficiency reduced mature cDC1 numbers in BM, spleen, lung, and intestine and Wnt4 directly induced of pJNK activation and cDC1 commitment. CD11cCreWnt4flox/flox mice lacked IRF8/cJun complex stabilization and had increased basal numbers of cDC2, group 2 innate lymphoid cells ILC2 and increased resistance to the parasite Nippostrongylus brasiliensis compared to CD11cCre controls. These data reveal a novel role for Wnt4 in DC commitment and pathogen-specific immunity.
Isolation of a 31 kDa protein in Angiostrongylus cantonensis from Hawaii and its comparative diagnostic efficacy to the 31 kDa protein from Thailand and crude antigen from Hawaii
Authors: Jarvi Susan Irene , Snook Kirsten Anna , Cannoles Kirsten , Quarta Stefano , Eamsobhana Praphathip , Shepherd Laura , Tagami Yaeko , Kaluna Lisa , Howe Kathleen , Jacquier Steven , Niebuhr Christopher , Bicakci Dehiz
Published: January 2023

The zoonotic metastrongylid nematode Angiostrongylus cantonensis is the primary cause of eosinophilic meningitis worldwide. East Hawaii Island is considered the US epicenter of the disease, also known as rat lungworm, and presentation is often severe. In 2015 we collected 435 blood samples and questionnaires from human volunteers for a pilot study on the prevalence of A. cantonensis in East Hawaii Island. Serum was tested by ELISA with crude antigen from Hawaii nematodes and by dot-blot ELISA with 31 kDa proteins isolated from Thailand nematodes. In the current study, consent was obtained from 199 of the original participants for the use of their previously collected samples. Nematodes were collected from the lungs and pulmonary arteries of rats in the course of animal studies, and SDS-PAGE gels were used to isolate the Hawaii 31 kDa proteins. The proteins were quantified with size determination by bioanalysis. The three different serological methods were compared. Of volunteers who defined themselves as definitively diagnosed by a clinician (n = 12), 40% were positive by Hawaii (HI) 31 kDa ELISA and reported being infected in or after 2004. Of those self-reported as previously infected with A. cantonensis (n=19), 31.5% were positive by HI 31 kDa ELISA; those who self-reported as uninfected (n=124), 22% tested positive by HI 31 kDa ELISA. The HI 31 kDa proteins showed an overall seroprevalence of 25% in the population, while the Thailand isolate of 31 kDa showed 30% and the HI crude antigen ELISA showed 22% prevalence, demonstrating that these antigens may have diagnostic efficacy.
Adherence of local isolates of Candida species to vaginal epithelial cells of apparently healthy and HIV-infected women in Nigeria, and pro-inflammatory cytokines response in a mouse model
Authors: Emele Felix Emele , Anieto Ezinne Chinenye
Published: January 2023

Background

Candida albicans (CA) has long been considered as the commonest cause of vulvovaginal candidiasis (VVC), while non-albicans Candida species (NAC) are mainly involved in opportunistic infections, particularly in HIV/AIDS. A prerequisite for colonization and eventual invasion of vaginal mucosa is the ability of Candida to adhere to vaginal epithelial cells (VECs); subsequent production of proinflammatory cytokines, such as IL-1β, IFN-γ, and IL-17, plays key roles in antifungal activity in the mucosa. Recent reports suggest increasing importance of non-albicans Candida species in VVC, in our region. This raised the need to compare the capacity of the local isolates of CA and NAC to adhere to VECs, and to elicit key inflammatory cytokines in mouse model.

Materials and Methods

Vaginal Epithelial Cells were collected from apparently healthy non-pregnant women, as well as those having HIV infection (n=16). The VECs were exposed to 12 local isolates of CA and NAC, in adherence assay. Mice were intra-vaginally inoculated with the Candida species and assayed for IL-1β, IL-17 and IFN-γ after 14 days.

Results

More NAC yeasts adhered to VECs of HIV infected women (Mean value = 5.6±3.8) than CA (2.7±3.3). Mean adherence of Candida to VECs from other sources were: HIV infected women with VVC (NAC = 6.2±4.7; CA = 5.1±1.7); apparently healthy women (CA = 2.90±3.40; NAC = 2.8±2.0). Mean levels (ng/ml) of IL-17 produced were: NAC (0.16±0.01) and CA (0.13±0.01).

Conclusion

Results tend to suggest that NAC is now as important as CA in VVC, and that pre-sensitization of VECs (of HIV+ women) increased Candida adherence. Depletion of IL-17 producing CD4+ cells could partly explain the preponderance of NAC in HIV/AIDS fungal opportunistic infections.
Aging impairs the cellular immune response to acute C. difficile infection
Authors: Close Lisa Abernathy , Dieterle Michael , Young Vincent
Published: January 2023

Clostridium difficile (C. difficile) is a spore-forming bacterium that produces enterotoxins that damage the intestinal epithelium. The incidence of C. difficile infection (CDI) increased significantly over the last two decades, particularly among those ≥65 years of age. The objective of this study was to determine the role of age in immune responses to C. difficile infection in mice, utilizing strains of varying virulence. We have shown that mice infected with C. difficile strain VPI 10463 (VPI) exhibit more severe disease compared to C. difficile strain 630. Thus, to examine an age-related effect on immune responses in CDI, young mice (2–3 months old) and aged mice (22–24 months old) were rendered susceptible with cefoperazone and then infected with either C. difficile strain 630 or VPI. Cecum and colon were harvested during peak disease severity (day 2–4 post-infection) and processed to assess intestinal immune cell profile by flow cytometry. We found that CD45+ immune cell infiltration occurs early in the cecum of mice infected with VPI, but not in 630, regardless of age. Of the CD45+ immune cells in the cecum and colon of VPI-infected mice, the majority were CD11b+ myeloid cells. While young mice infected with C. difficile strain VPI showed a robust increase in cecal eosinophils, there was a significant lack of an eosinophil response in aged counterparts. In summary, C. difficile strain 630 did not elicit an early cellular intestinal immune response and this was independent of age. Furthermore, aged mice demonstrated a significantly decreased local cellular immune response to C. difficile VPI 10463 infection compared to young mice, suggesting a role for age-associated immune responses in CDI outcomes in older patient populations.
NKp46+ NK cells regulate brain CD4+ T cells to promote chronic Toxoplasma gondii infection
Authors: Gigley Jason P. , Ivanova Daria L , Denton Stephen L
Published: January 2023

Reactivation of chronic Toxoplasma gondii (T. gondii) infection and toxoplasmic encephalitis is a major health concern in immune compromised people. Dysregulated T-cell responses are thought to promote chronic Toxoplasmosis. The non-T-cell factors that contribute to T cell dysregulation and chronic T. gondii infection are unclear. We have discovered that NKp46+ NK cells paradoxically increase the frequency and number of IFNγ+TNFα+CD4+ T-cells in the brain in during chronic T. gondii infection. Depletion of NK cells resulted in a decrease in polyfunctional brain CD4+ T-cells and an increase in IFNγ+Grzb+CD8+ T-cell responses in the brain. This decrease in CD4+ T-cell and increase in CD8+ T-cell responses correlated with better survival of chronically infected mice. This result suggests that NKp46+ NK cells drive an NK cell-dependent CD4+ T cell response that is detrimental to the CD8+ T cell response important for controlling chronic T. gondii infection. In support of this possibility, adoptive transfer of NK cell-dependent CD4+ T cells into chronically infected mice reduced polyfunctional CD8+ T-cell responses in the brain while CD4+ T cells generated in the absence of NK cells during chronic T. gondii infection did not. NKp46+ NK cells were not found in brain tissue, but appear to localize to the vasculature of the brain and periphery during chronic T. gondii infection. Surprisingly, we observe that NKp46+ NK cells increase their expression of MHC Class II. Our results lead us to hypothesize that during chronic T. gondii infection NKp46+ NK cells acquire the ability to prime CD4+ T-cell responses via MHC Class II expression resulting in decreased polyfunctional CD8 T cell responses to promote parasite persistence.
THE IL-12- AND IL-23-DEPENDENT NK-CELL RESPONSE IS ESSENTIAL FOR PROTECTIVE IMMUNITY AGAINST SECONDARY TOXOPLASMA GONDII INFECTION
Authors: Denton Stephen L , Ivanova Daria L , Mundhenke Tiffany , Gigley Jason P.
Published: January 2023

Natural Killer (NK) cells can develop memory-like features and contribute to long-term immunity in mice and humans. NK cells are critical for protection against acute T. gondii infection. However, whether they contribute to long-term immunity in response to this parasite is unknown. We used a vaccine challenge model of parasite infection to address this question and to define the mechanism by which NK cells are activated during secondary parasite infection. We found NK cells were required for control of secondary infection. NK cells increased in number at the infection site, became cytotoxic and produced IFNγ. Adoptive transfer and NK-cell fate mapping revealed that T. gondii-experienced NK cells were not intrinsically different from naïve NK cells with respect to their long-term persistence and ability to protect. Thus, they did not develop memory-like characteristics. Instead, a cell-extrinsic mechanism may control protective NK-cell responses during secondary infection. To test the involvement of a cell-extrinsic mechanism, we used anti-IL-12p70 depletion and IL-12p35−/− mice and found that the secondary NK-cell response was not fully dependent on IL-12. IL-23 depletion with anti-IL-23p19 in vivo significantly reduced the secondary NK-cell response, suggesting that both IL-12 and IL-23 were involved. Anti-IL-12p40 treatment, which blocks both IL-12 and IL-23, eliminated the protective secondary NK-cell response, supporting this hypothesis. Our results define a previously unknown protective role for NK cells during secondary T. gondii infection that is dependent on both IL-12 and IL-23.
Innate and adaptive immune interactions in Coccidioides infection
Authors: Hoyer Katrina K , Diep Anh
Published: January 2023

Protective immunity against the fungal pathogen Coccidioides requires specific T helper cellular responses. Mouse infection and vaccine studies have defined CD4 T helper (Th)1 and Th17 cells in the resolution of infection and in effective protection, while patients with chronic Coccidioides infection demonstrate reduced cellular responses. Several gaps in knowledge preclude our ability to develop new diagnostics and therapeutics for treating this infection. Little is known about the microenvironment in human lung tissue during infection, and the types of immune cells that promote effective control of the fungus within this specialized location. How variations on local immune populations alter disease severity and ability of the fungus to disseminate have not been explored. We recently identified several peripheral blood biomarkers during acute infection that distinguish pediatric patients that resolve infection from those that develop chronic disease. Patients with chronic disease had reduced Th17 and elevated Treg frequencies compared to patients that resolved disease. The inability to clear Coccidioides infection may be a result of elevated Treg frequency and functional capacity, or of skewed effector differentiation. We use mouse Coccidioides infection and in vitro culture assays to uncover the host immune players that promote effective immunity, and to identify fungal morphology changes and fungal burden differences at the innate and adaptive immune interface.
ICOS expression is not required for memory T cell formation or maintenance, but does promote Tfh cell differentiation and the humoral response after secondary Plasmodium chabaudi chabaudi AS infection
Authors: Latham Leah E , Wikenheiser Daniel J , Stumhofer Jason S
Published: January 2023

In addition to promoting follicular helper T (Tfh) cell development, ICOS is implicated in memory T cell formation. We have shown in the P. chabaudi rodent model of malaria that the absence of ICOS leads to a persistent non-resolving infection due to a defect in Tfh cell maturation that prevents the formation of a germinal center (GC) response. Here we show that memory T cell formation and maintenance is not impaired in the absence of ICOS. The presence of CXCR5-expressing central memory T (TCM) cells in Icos−/− mice suggests that they are capable of differentiating into Tfh cells and supporting the humoral response after challenge. However, the formation of Tfh cells was impaired in Icos−/− mice after challenge. Similar to the primary infection, drug-cleared Icos−/− mice displayed a relapsing parasitemia that occurred more frequently and with higher peaks compared to wild-type mice, which was associated with a diminished and delayed parasite-specific antibody response and the absence of GCs. Surprisingly, GCs that were present in the spleen of wild-type mice pre-challenge dispersed shortly after challenge, but were apparent again by day 35 post-challenge. Adoptive transfer of TCM cells from wild-type and Icos−/− mice into Tcrβ−/− mice to directly evaluate the ability of TCM cells to differentiate into Tfh cells revealed that CXCR5+ and CXCR5− TCM cells from wild-type mice could develop into Tfh cells in the presence or absence of memory B cells. However, TCM cells from Icos−/− mice were incapable of differentiating into Tfh cells, although they could support GC-independent Ab production. Thus, ICOS expression is not required for memory T cell formation, but is necessary for the differentiation of TCM cells into Tfh cells after challenge.
Tuberculosis boosts HIV-1 production by macrophages through IL-10/STAT3 dependent tunneling nanotube formation
Authors: Lugo Geanncarlo , Souriant Shanti , Balboa Luciana , Dupont Maeva , Pingris Karine , Kviatcovsky Denise , Cougoule Celine , Lastrucci Claire , Bah Aicha , Gasser Roman , Poincloux Renaud , Raynaud-Messina Brigitte , Al Saati Talal , Inwentarz Sandra , Poggi Susana , Moraña Eduardo Jose , González-Montaner Pablo , Corti Marcelo , Lagane Bernard , Vergne Isabelle , Allers Carolina , Kaushal Deepak , Kuroda Marcelo , del Carmen Sasiain Maria , Neyrolles Olivier , Maridonneau-Parini Isabelle , Verollet Christel
Published: January 2023

The tuberculosis (TB) bacillus, Mycobacterium tuberculosis (Mtb) and HIV-1 are known to act synergistically, however, the mechanisms by which Mtb exacerbates HIV-1 pathogenesis are not well known. Using in vitro and ex vivo cell culture system, we show that human M(IL-10) anti-inflammatory macrophages, present in TB-associated microenvironments, produced high levels of HIV-1. In vivo, M(IL-10) macrophages were expanded in lungs of co-infected non-human primates, their number correlated with disease severity, and markers for these cells (soluble CD163 and MerTK) accumulated in the blood of co-infected patients. These M(IL-10) macrophages formed direct cell-to-cell bridges, which we identified as tunneling nanotubes (TNTs) involved in viral transfer. TNT formation required the IL-10/STAT3 signaling pathway, and targeted inhibition of TNTs substantially reduced the enhanced HIV-1 cell-to-cell transfer and overproduction in M(IL-10) macrophages. Our study reveals that TNTs facilitate viral transfer and amplification, promoting TNT formation as a mechanism to be explored in TB/AIDS potential therapeutics.
Induction of inflammatory responses in splenocytes by exosomes released from intestinal epithelial cells following Cryptosporidium parvum infection
Authors: Chen Xian-Ming , Wang Yang , Shen Yujuan , Liu Hua , Yin Jianhai , Zhang Xin-Tian , Gong Ai-Yu , Chen Xiqiang , Chen Siyi , Mathy Nicholas W , Cao Jianping
Published: January 2023

Cryptosporidium, a protozoan parasite that infects the gastrointestinal epithelium and other mucosal surfaces in humans and animals, is an important opportunistic pathogen in AIDS patients and one of the most common enteric pathogens affecting young children in developing regions. We previously demonstrated that C. parvum infection stimulates host epithelial cells to release exosomes and these released exosomes shuttle several antimicrobial peptides to carry out anti-C. parvum activity. Murine intestinal epithelial (IEC4.1) cells and neonatal mice were applied for infection. Primary splenocytes were exposed to exosomes isolated from infected cells or un-infected controls. Inflammatory response in splenocytes were measured. We detected upregulation of inflammatory genes in the liver and spleen following C. parvum intestinal infection in neonatal mice. Interestingly, exosomes released from intestinal epithelial cells following C. parvum infection could activate the nuclear factor kappa B signaling pathway and trigger inflammatory gene transcription in isolated primary splenocytes. Several epithelial cell-derived proteins and a subset of parasite RNAs were detected in the exosomes released from C. parvum-infected intestinal epithelial cells. Shuttling of these effector molecules, including the high mobility group box 1 protein, was involved in the induction of inflammatory responses in splenocytes induced by the released exosomes from infected cells. Our data indicate that exosomes released from intestinal epithelial cells upon C. parvum infection can activate immune cells through shuttling various effector molecules, a process that may be relevant to host systemic responses to Cryptosporidium infection.
Microbiota-derived butyrate limits the autoimmune response by promoting follicular regulatory T cells
Authors: Takahashi Daisuke , Hoshina Naomi , Clarke Julie M. , Hase Koji
Published: January 2023

Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disorder with a high prevalence, especially in industrialized countries. Dysbiosis of the intestinal microbiota has been observed in RA patients. For instance, new-onset untreated RA is associated with the expansion of Prevotellaceae and the under representation of the Clostridiumcluster XIVa, including Lachnospiraceae, which are major butyrate producers in the gut, although the pathological relevance has remained obscure. Here, we report that microbiota fermentation product butyrate controls the development of autoimmune arthritis in the host. We found that systemic autoantigen immunization causes hyperplasia of gut-associated lymphoid tissues by amplifying the germinal center (GC) reaction. Butyrate mitigated these pathological events by increasing Bcl-6+CXCR5+CD25−Foxp3+ follicular regulatory T (TFR) cells. Butyrate directly induced the differentiation of functional TFR cells by enhancing histone acetylation in TFR cell marker genes. The adoptive transfer of butyrate-induced TFR cells prevented autoimmune arthritis in mice. Notably, TFR cells induced by butyrate suppressed the development of autoreactive follicular helper T (TFH) and GCB cells and their migration from GALT into articulation-draining lymph nodes (DLNs). Accordingly, microbiota-derived butyrate serves as an environmental cue to enhance TFR cells which suppress the GC reaction and autoantibody production in the systemic lymphoid tissue, eventually ameliorating autoimmune arthritis. Our findings provide mechanistic insights into the link between the gut microbiota and RA risk.
Functional characterization of the enteric animal virome
Authors: Dallari Simone , Heaney Thomas , Rosas Adriana , Cadwell Ken
Published: January 2023

The enteric virome includes viruses that infect eukaryotic cells in the gut and is one constituent of the mammalian microbiome. Although best known for causing acute diarrheal disease, many of these viruses are detected in asymptomatic individuals. The immune consequence of harboring these viruses is unclear. We recently demonstrated that norovirus infection promotes the development of the mucosal immune system in mice in a manner similar to symbiotic bacteria, and can protect against models of chemical and microbial injury. These results are reminiscent of findings from the microbiome field demonstrating that intestinal colonization by individual bacterial species directs the differentiation of the immune system. Here, we examined the extent to which the eukaryotic enteric virome contributes to the state of the host immune system. Examination of 10 enteric DNA and RNA viruses, representing 6 viral families, revealed that many establish prolonged infection in the absence of disease. To evaluate the immunological impact of these viral infections that occurs independently of other members of the microbiome, we performed a comprehensive flow cytometry analysis of several organs following infection of germ-free mice that assessed over 20 immune cell subsets and their capacity for cytokine production. We found profound effects of enteric viral infection in all the tissues tested, showing strain-specific responses, such as norovirus-induced expansion of type 1 regulatory T cells, and commonly shared ones, such as the increased potential of colonic T cells to produce Il-22. Taken together, these data demonstrate that enteric virome members contribute to the immune status of the host.
IL-6, IL-8 drive LAG3/PD1 immune suppression on effector and naïve, peripheral blood CD8+ T cells in cancer patients
Authors: Somasundaram Ashwin , Cillo Anthony , Oliveri Lauren , Herman James , Kirkwood John , Ferris Robert L , Bruno Tullia Carmela , Vignali Dario
Published: January 2023

Cancer patients off therapy and with normal white blood cell counts are often at greater risk for infections, immune dysregulation, progressive disease or reactivation of viral infections. However, the exact mechanism of this systemic immunosuppression in cancer patients is not fully understood. We hypothesize that cancer patients may have systemic immune suppression via cytokine-driven IR expression in all CD8+ T cells subsets, including naïve cells.

PBL were obtained from healthy donors and treatment-naïve NSCLC, HNSCC, and melanoma patients. IR (i.e. LAG3, PD1, CTLA4, etc) expression was assessed on CD8+ T cells, CD4+ T cells, and regulatory T cells. Plasma cytokine concentrations were compared by Luminex. Autologous micro-stimulation assays were performed on peripheral CD8+ or CD4+ T cells with antigen presenting cells plus or minus IR blockade.

CD8+ T cells, including naive CD8+ T cells, from cancer patient PBL contain elevated total LAG3 expression which correlated with worse state, worse survival, and elevated expression of other IRs. Further, CD8+ T cells from these patients had decreased proliferation, which was rescued with the addition of anti-LAG3 or anti-PD1. Plasma from these patients had significantly elevated levels of cytokines that can signal via STAT3 (i.e. IL-6, IL-8), which were independently found to increase total IR expression in healthy donor, naïve CD8+ T cells.

Patients with cancer have systemic elevations IL-6 and IL-8 leading to increased IR expression in naïve, peripheral CD8+ T cells making them poised for exhaustion even before TCR binding. These findings suggest that cytokine combined with IR blockade may play a role in reversing PD1 blockade resistance.
Avian IgY antibodies recognize novel Dengue NS1 epitopes with the ability to neutralize Dengue 2 virus infection in vitro and in vivo
Authors: O’Donnell Kyle Lee , Espinosa Diego , Nilles Matthew L , Harris Eva , Bradley David
Published: January 2023

Dengue virus (DENV) causes 96 million infections worldwide resulting in 500,000 cases of Dengue Hemorrhagic Fever (DHF) and 22,000 deaths. The severity of DENV infection is greatly enhanced by antibody dependent enhancement (ADE); increased viral load due to increased monocyte opsonization by non-neutralizing low avidity Abs from a previous infection with a different DENV serotype or secondary flavivirus infection. We have previously demonstrated that polyclonal avian anti-DENV-2 IgY ameliorates DENV infection in mice without inducing ADE. The whole polyclonal anti-DENV-2 IgY preparation contained distinct populations of IgY that recognized numerous epitopes of the envelope, membrane, and non-structural (NS) DENV proteins. We report here that two purified pools of anti-DENV-2 IgY with specificity for two different regions of NS1 had a neutralization capacity equivalent to the whole polyclonal anti-DENV-2 IgY preparation in vitro. We assayed each pool of NS1-specific IgY for the potential to induce ADE and demonstrated that neither pool induced ADE when cells were treated with IgY and challenged with a flavivirus. To determine if these NS1 specific IgY achieved neutralization via reducing vascular leakage, we utilized a trans-endothelial electrical resistance (TEER) assay. Interestingly, treatment with DENV NS1-specific IgY did not reduced vascular leakage in our model system. Final to demonstrate the therapeutic potential of NS1 specific IgY we prophylactically treated IFNAR−/− mice with 150 μg of NS1 specific IgY and saw protective efficacy up to 100% in a lethal dengue challenge. We hypothesize that the neutralizing effect of the NS1 specific IgY is via the antibody disrupting the stability of the viral replication complex.