Gut-associated Lymphoid Tissue Research Articles

Potential Health Benefits of Functional Foods in Boosting Immunity and Relationship between Immunosuppressant Diseases and Severity of Viral Infections-COVID-19 – A Review Article

COVID-19 pandemic has been a global challenge to all the nations and has really affected the sense of normalcy in the lives of People. It has been a sense of awakening that has made people realize the role of immunotherapy in fighting against such situations. It has been observed that the strong gut-associated lymphoid tissue (GALT) which accounts as a dominant role in the immunity has a very important role to play in strengthening the immune system. Dysbiosis could be a major cause of many health disorders and immune suppressants diseases. It was strongly observed that patients with compromised immune system badly affected in COVID-19. In this review, we are trying to find out the association between functional foods such as Probiotics, Prebiotics, and various other Nutraceuticals in strengthening the immune system and its potential benefit to act as preventive medicine in curbing down incidence or management of COVID-19.

The injuries of spleen and intestinal immune system induced by 2-Gy 60Co γ-ray whole-body irradiation

Purpose The aim of the present study was to investigate the injuries of spleen and intestinal immune system induced by 2 Gy 60Co γ ray in mice. Materials and methods A total of 120 Balb/c mice were randomly divided into two groups: blank control (Ctrl) and model (IR). The IR mice were exposed to a single dose of total body irradiation (2 Gy, dose rate: 1 Gy/min) and sacrificed on 1st, 3rd, 7th, 14th and 21st day after irradiation. The indicators including general observations and body weight, the changes in peripheral hemogram, spleen index, histopathology examination and lymphocyte subsets of spleen. As well as the count and subsets of lymphocyte in gut-associated lymphoid tissue. Results Compared with the Ctrl group, the body weight, spleen index, peripheral blood cell and splenocyte amounts, intraepithelial lymphocytes number decreased significantly after exposure, accompanied by a notable decreased count of lymphocytes in Peyer’s patch and mesenteric lymph nodes. Moreover, ionizing radiation also broke the balance of CD4+/CD8+ and increased the Treg proportion in spleen, which then triggered immune imbalance and immunosuppression. In general, the spleen injuries occurred on 1st day after exposure, worse on 3rd day, and were relieved on 7th day. The intestinal immune injuries were observed on 1st day, and attenuated on 3rd day. On 21st day after exposure, the spleen volume and index have returned to normal, except for the distribution of lymphocyte subpopulations. Furthermore, all indicators of gut-associated lymphoid tissue, except for mesenteric lymph nodes lymphocyte count, had returned to normal levels on 21st day. Conclusion In conclusion, our data showed the injuries of spleen and intestinal immune system induced by 2 Gy 60Co γ ray whole-body irradiation. These findings may provide the bases for further radiation protection in the immunity.

No evidence of uptake or propagation of reindeer CWD prions in environmentally exposed sheep

BackgroundChronic wasting disease (CWD) is a prion disease of cervids first reported in North America in the 1960s. In Europe, CWD was first diagnosed in 2016 in a wild reindeer in Norway. Detection of two more cases in the same mountain area led to the complete culling of this partially confined reindeer population of about 2400 animals. A total of 19 CWD positive animals were identified. The affected area is extensively used for the grazing of sheep during summers. There are many mineral licks intended for sheep in the area, but these have also been used by reindeer. This overlap in area use raised concerns for cross-species prion transmission between reindeer and sheep. In this study, we have used global positioning system (GPS) data from sheep and reindeer, including tracking one of the CWD positive reindeer, to investigate spatial and time-relevant overlaps between these two species. Since prions can accumulate in lymphoid follicles following oral uptake, samples of gut-associated lymphoid tissue (GALT) from 425 lambs and 78 adult sheep, which had grazed in the region during the relevant timeframe, were analyzed for the presence of prions. The recto-anal mucosa associated lymphoid tissue (RAMALT) from all the animals were examined by histology, immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA), and the ileal Peyer's patch (IPP) from a subsample of 37 lambs were examined by histology and IHC, for the detection of prions.ResultsGPS data showed an overlap in area use between the infected reindeer herd and the sheep. In addition, the GPS positions of an infected reindeer and some of the sampled sheep showed temporospatial overlap. No prions were detected in the GALT of the investigated sheep even though the mean lymphoid follicle number in RAMALT and IPP samples were high.ConclusionThe absence of prions in the GALT of sheep that have shared pasture with CWD-infected reindeer, may suggest that transmission of this novel CWD strain to sheep does not easily occur under the conditions found in these mountains. We document that the lymphoid follicle rich RAMALT could be a useful tool to screen for prions in sheep.

Nano-strategies as Oral Drug Delivery Platforms for Treatment of Cancer: Challenges and Future Perspectives.

Oral drug administration is the oldest and widely used method for drug administration. The objectives behind developing an oral drug delivery for the treatment of cancer are to achieve low cost treatment by utilizing novel techniques to target cancer through gut-associated lymphoid tissue (GALT) and to enhance patient comfort and compliance through a hospital-free treatment leading to "Chemotherapy at Home." Unfortunately, due to the physiological environment of the GIT and physicochemical properties of drug candidate, the efficacy of oral drug delivery methods is limited in the treatment of cancer. Due to their low hydrophilicity, high P-gp efflux and restricted intestinal permeability most of the anti-cancer drugs fail to achieve oral bioavailability. The review focuses on the efforts, challenges, opportunities and studies conducted by scientists worldwide on the oral administration of anticancer medications via nanocarriers such as liposomes, SLNs and dendrimers, because of their potential to overcome the epithelial barrier associated with GALT, as well as the applications of different polymers in targeting the cancer. The oral delivery can set newer horizons in cancer therapy to make it more patient friendly.

Cecal Patches Generate Abundant IgG2b-Bearing B Cells That Are Reactive to Commensal Microbiota.

Gut-associated lymphoid tissue (GALT), such as Peyer's patches (PPs), are key inductive sites that generate IgA+ B cells, mainly through germinal center (GC) responses. The generation of IgA+ B cells is promoted by the presence of gut microbiota and dietary antigens. However, the function of GALT in the large intestine, such as cecal patches (CePs) and colonic patches (CoPs), and their regulatory mechanisms remain largely unknown. In this study, we demonstrate that the CePs possess more IgG2b+ B cells and have fewer IgA+ B cells than those in PPs from BALB/c mice with normal gut microbiota. Gene expression analysis of postswitched transcripts supported the differential expression of dominant antibody isotypes in B cells in GALT. Germ-free (GF) mice showed diminished GC B cells and had few IgA+ or IgG2b+ switched B cells in both the small and large intestinal GALT. In contrast, myeloid differentiation factor 88- (MyD88-) deficient mice exhibited decreased GC B cells and presented with reduced numbers of IgG2b+ B cells in CePs but not in PPs. Using ex vivo cell culture, we showed that CePs have a greater capacity to produce total and microbiota-reactive IgG2b, in addition to microbiota-reactive IgA, than the PPs. In line with the frequency of GC B cells and IgG2b+ B cells in CePs, there was a decrease in the levels of microbiota-reactive IgG2b and IgA in the serum of GF and MyD88-deficient mice. These data suggest that CePs have a different antibody production profile compared to PPs. Furthermore, the innate immune signals derived from gut microbiota are crucial for generating the IgG2b antibodies in CePs.

FC050: Nefecon® Selectively Modifies the Composition of Circulating Immune Complexes in IGA Nephropathy

Abstract BACKGROUND AND AIMS IgA nephropathy (IgAN) is characterized by the mesangial deposition of IgA-containing immune complexes (IgA-IC). There is, however, no clear association between extent of mesangial IgA-IC deposition and disease severity in IgAN, suggesting that the composition of the IgA-IC, rather than the actual amount, may be critical in determining pathogenic consequences. The NEFIGAN trial (NCT01738035) assessed the safety and efficacy of a novel targeted-release formulation of budesonide (Nefecon®), designed to deliver the drug to the gut-associated lymphoid tissue (GALT)-rich distal ileum in patients with IgAN. The trial comprised a 6-month run-in, a 9-month treatment and a 3-month follow-up phase. A total of 48 patients received Nefecon® 16 mg/day, 51 patients received Nefecon® 8 mg/day and 50 patients received placebo. The key result of the study was that Nefecon® 16 mg/day, added to optimized renin–angiotensin system blockade, reduced proteinuria and stabilized estimated glomerular filtration rate in patients with IgAN. These findings have now been replicated in the NefIgArd study, which released data in 2021. Here, we report the composition of IgA-IC in the serum of patients treated with Nefecon® 16 mg/day or placebo in the NEFIGAN trial, assessed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). This study was supported by a grant from Calliditas Therapeutics AB. METHOD Using Jacalin Agarose and elution with 0.1 M d-galactose, IgA-ICs were affinity-purified from serum of patients who received placebo (n=18) or Nefecon® 16 mg/day (n=18) at the start of treatment (SOT) and end of treatment (EOT). All protein samples were reduced and alkylated prior to digestion with trypsin, dried using a Savant SpeedVac (Thermo Fisher Scientific, UK) and dissolved in 40 µL of 0.1% formic acid. Each sample, spiked with iRT standard peptides (Biognosys, Switzerland), was loaded onto an Evotip (Evosep Biosystems, Denmark) column. Mass spectrometry analyses were performed on the Evosep One (Evosep) platform, coupled to the TIMS TOF Pro (Bruker, US). For protein identification and quantification, raw data were processed with FragPipe (V16), based on at least two peptides per protein. The protein intensities thus generated were Log_2-transformed and internally normalized using IGHA1 protein as standard. For each patient, the normalized protein amount at SOT was subtracted from the amount at EOT. A probabilistic dropout model was applied to the data to estimate fold changes between treatment and placebo groups at EOT. The proteins were ranked using t-statistic, and a gene set enrichment analysis was performed to determine the gene sets significantly affected by Nefecon® 16 mg/day versus placebo. RESULTS Gene ontology analysis revealed that treatment with Nefecon® 16 mg/day led to a significant protein enrichment in IgA-IC associated with pathways driving biological processes involved in the negative regulation of gene expression and transcription, gene silencing and epigenetics. In parallel, treatment with Nefecon® was also associated with an enrichment of proteins associated with extracellular vesicle and exosome formation. CONCLUSION For the first time, we show that it is possible to pharmacologically modify the composition of circulating IgA-IC in IgAN. Treatment with Nefecon® 16 mg/day for 9 months selectively altered the protein composition of IgA-IC in a way that is likely to have significant consequences on the downstream mesangial response to IgA-IC accumulation; this may, in part, explain the antiproteinuric effect of this approach and the longer-term kidney function protection that has been reported in both the NEFIGAN and NefIgArd clinical trials. Studies are ongoing to precisely delineate the impact of these compositional changes on the pathogenicity of IgA-IC in IgAN.

Coronin 1A is Uniquely Expressed in the Human Follicle Associated Epithelium and is Required For Human M Cell Maturation and Function

IntroductionMicrofold (M) cells are specialized epithelial cells that play an integral role in immune surveillance of the gut luminal environment. They functionally act as gatekeepers within the follicle‐associated epithelium (FAE) of Peyer’s patches to regulate antigen delivery to immune cells of gut‐associated lymphoid tissue. Studies have associated damage in the FAE with chronic inflammatory conditions, such as IBD; however, studies designed to understand how human M cells contribute to disease onset and/or pathogenesis under pathogenic conditions remain incomplete. OBJECTIVE: Since M cells are differentiated from Lgr5+ intestinal stem cells, we proposed to utilize human enteroids to determine the factors that regulate human M cell differentiation and function. Quantitative proteomics analysis of human enteroids differentiated to express M cells revealed enriched expression of coronin 1A, an actin‐remodeling protein involved in phagocytosis and expressed immune cells. Since M cells engulf luminal gut antigens via phagocytosis, we hypothesized that coronin 1A is uniquely expressed in the human FAE and plays a role in gut antigen uptake and transcytosis in human M cells.MethodsHuman ileal enteroids derived from biopsies obtained from healthy donors were grown as monolayers and differentiated (no Wnt3a, R‐spondin‐1) for at least 5 days. To express M cells, cultures were exposed to RANKL and TNF during differentiation. Human expression of coronin 1A was validated by immunoblot and confocal microscopy of ileal biopsies obtained from healthy donors. Stable knockdown (KD) of coronin 1A in human enteroids was achieved by shRNA lentivirus and puromycin selection. M cell monolayers, expressing or lacking coronin 1A, were exposed apically to commensal (HS) and pathogenic strains of E. coli (EPEC, AIEC) to determine the contribution of gut microbes to stimulate M cell transcytosis, as measured by uptake of fluorescent polystyrene latex beads and immunoglobulins.ResultsCoronin 1A was expressed in human enteroid monolayers differentiated to express M cells, but not in monolayers differentiated to mimic the villus epithelium. Similar results were obtained in biopsy sections of the human ileum with coronin 1A detected in the FAE, but not in crypt or villus epithelia. This staining pattern was unique for human, as coronin 1A expression in the mouse ileum was confined to lamina propria and lymphoid follicle immune cells. Coronin 1A KD resulted in decreased expression of glycoprotein 2, a marker of mature M cells, while other markers of the FAE and M cells, including CD14, ICAM1, Spi‐B, SOX8, and FcRN remained unchanged. Coronin 1A KD also resulted in impaired uptake/transcytosis of IgG (via FcRN) as well as pathogen‐stimulated uptake/transcytosis of polystyrene beads by human M cells.ConclusionsThe studies show: (1) human enteroids can be differentiated to model coronin 1A expression in the intact human FAE; (2) coronin 1A expression is necessary for terminal differentiation and function of human M cells; and (3) coronin 1A is required for pathogen‐stimulated uptake and transcytosis of various types of luminal gut cargo. We conclude that M cell‐expressing enteroid monolayers can be used as a relevant human model to further interrogate tropism for host‐pathogen interactions, mechanisms of luminal surveillance, and response to oral vaccine therapies.

The development of a gut-specific immune response to an oral antigen is dependent on α4β7 mediated lymphocyte trafficking

Abstract Objective Vedolizumab has been shown to reduce the antibody response to an oral cholera, but not intramuscular hepatitis B vaccine in healthy subjects (Wyant, Gut 2015). The generation of an immune response to an oral antigen (Ag) begins at inductive sites such as Peyer’s patches (PP) followed by cell migration to effector sites in the gut. The integrin α4β7 is known to play a major role in cell trafficking to gut-associated lymphoid tissues. This study assessed whether MT-102, a potent and selective small molecule α4β7 inhibitor has similar effects on immunizations to those reported in the clinic for vedolizumab. Methods An in vivo immunization model system was established to determine the effects of MT-102, dosed via osmotic minipump, on the development of a gut-specific immune response. Mice were orally immunized with cholera toxin (CTX), and anti-CTX antibodies were quantified in fecal and plasma samples. The impact of MT-102 on lymphocytes within the PP of immunized mice was determined by flow cytometry. Results MT-102 delayed the production of anti-CTX IgA and IgG antibodies as measured in fecal samples without perturbing total gut IgA levels. Furthermore, MT-102 also inhibited the levels of Ag-specific antibodies in the plasma following repeated CTX oral immunizations. Blocking α4β7 induced a decrease in the frequency of B cells in the PP of treated mice compared to the control group. Conclusion α4β7-specific inhibitor, MT-102, effectively diminished the mucosal antibody response to an orally delivered Ag in mice. These results are consistent with what has been reported clinically in vedolizumab-treated subjects and thus establishes that potent small molecule inhibitors of α4β7 can replicate the pharmacology of an antibody.

Gastrointestinal Manifestations of Immunodeficiency: Imaging Spectrum.

There is a wide spectrum of hereditary and acquired immunodeficiency disorders that are characterized by specific abnormalities involving a plethora of humoral, cellular, and phagocytic immunologic pathways. These include distinctive primary immunodeficiency syndromes due to characteristic genetic defects and secondary immunodeficiency syndromes, such as AIDS from HIV infection and therapy-related immunosuppression in patients with cancers or a solid organ or stem cell transplant. The gut mucosa and gut-associated lymphoid tissue (the largest lymphoid organ in the body), along with diverse commensal microbiota, play complex and critical roles in development and modulation of the immune system. Thus, myriad gastrointestinal (GI) symptoms are common in immunocompromised patients and may be due to inflammatory conditions (graft versus host disease, neutropenic enterocolitis, or HIV-related proctocolitis), opportunistic infections (viral, bacterial, fungal, or protozoal), or malignancies (Kaposi sarcoma, lymphoma, posttransplant lymphoproliferative disorder, or anal cancer). GI tract involvement in immunodeficient patients contributes to significant morbidity and mortality. Along with endoscopy and histopathologic evaluation, imaging plays an integral role in detection, localization, characterization, and distinction of GI tract manifestations of various immunodeficiency syndromes and their complications. Select disorders demonstrate characteristic findings at fluoroscopy, CT, US, and MRI that permit timely and accurate diagnosis. While neutropenic enterocolitis affects the terminal ileum and right colon and occurs in patients receiving chemotherapy for hematologic malignancies, Kaposi sarcoma commonly manifests as bull's-eye lesions in the stomach and duodenum. Imaging is invaluable in treatment follow-up and long-term surveillance as well. Online supplemental material is available for this article. ©RSNA, 2022.

CXCR3 Expression Pattern on CD4+ T Cells and IP-10 Levels with Regard to the HIV-1 Reservoir in the Gut-Associated Lymphatic Tissue.

(1) Background: The gut-associated lymphatic tissue (GALT) represents the largest lymphoid organ, and is considered to be the largest HIV reservoir. The exact size of the GALT reservoir remains unclear. Several markers, such as the chemokine receptor CXCR3 and its pro-inflammatory ligand IP-10, have been proposed to define the size of HIV reservoirs in the peripheral blood (PB). However, little is known about the role of CXCR3 and IP-10 within the GALT. (2) Methods: We compared the CXCR3 expression, IP-10 levels, and cell-associated HIV DNA of distinct memory CD4+ T cell subsets from the terminal ileum (TI), PB and rectum (RE) of 18 HIV+ patients with antiretroviral therapy (ART), 6 HIV+ treatment-naive patients and 16 healthy controls. (3) Results: While the relative distributions of CD4+ T cell subsets were similar in PB, TI and RE, HIV DNA and CXCR3 expression were markedly increased and IP-10 levels were decreased in TI when compared to PB. No significant correlation was found between the CXCR3 expression and memory CD4+ T cell subsets, IP-10 levels and the HIV DNA amounts measured in PB, TI or RE. (4) Conclusions: During a chronic HIV-1 infection, neither CXCR3 nor IP-10 are indicative of the size of the viral reservoir in the GALT (TI and RE).

Indole-3-propionic acid alleviates ischemic brain injury in a mouse middle cerebral artery occlusion model

Increasing evidence highlights the importance of gut microbiota and its metabolites as an environmental factor affecting ischemic stroke. However, the role of microbial indole metabolites in ischemic stroke remains largely unknown. Here, we evaluated the effects and the underlying mechanism of indole-3-propionic acid (IPA) in a mouse model of acute middle cerebral artery occlusion (MCAO) and the mechanisms underlying these effects. We collected blood samples and evaluated serum indole derivatives levels using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS) in 8–10-week-old male C57 mice undergoing MCAO or sham. Intragastric IPA administration (400 μg/20 g/d) was performed in mice with MCAO, and its effects and mechanisms were assessed. We found that the serum IPA levels were significantly lower in mice with MCAO than in sham-treated subjects. 16S rRNA gene sequencing revealed that IPA treatment ameliorated the MCAO-induced alterations of the gut microbiome structure, specifically reshaping the microbial community composition in mice with MCAO to resemble that in the mice from the control group, with an increase in the abundance of probiotics and a decrease in the abundance of harmful bacteria. IPA repaired the integrity of the intestinal barrier and regulated the activities of regulatory T cells (Tregs) and Th17 cells in the gut-associated lymphoid tissue. Intragastric IPA administration effectively alleviated neuroinflammation, neurological impairment and brain infarction. Of note, Tregs in the IPA treatment group inhibited A1 reactive astrogliosis in vitro. The beneficial effects of IPA are thus mediated by the gut microbiota, which could enable the development of prebiotics for microbiome-based treatments for ischemic stroke.

The impact of the gut microbiota on T cell ontogeny in the thymus.

The intestinal microbiota is critical for the development of gut-associated lymphoid tissues, including Peyer's patches and mesenteric lymph nodes, and is instrumental in educating the local as well as systemic immune system. In addition, it also impacts the development and function of peripheral organs, such as liver, lung, and the brain, in health and disease. However, whether and how the intestinal microbiota has an impact on T cell ontogeny in the hymus remains largely unclear. Recently, the impact of molecules and metabolites derived from the intestinal microbiota on T cell ontogeny in the thymus has been investigated in more detail. In this review, we will discuss the recent findings in the emerging field of the gut-thymus axis and we will highlight the current questions and challenges in the field.

Two subsets of human marginal zone B cells resolved by global analysis of lymphoid tissues and blood.

B cells generate antibodies that are essential for immune protection, but their subgroups are poorly defined. Here, we perform undirected deep profiling of B cells in matched human lymphoid tissues from deceased transplant organ donors and blood. In addition to identifying unanticipated features of tissue-based B cell differentiation, we resolve two subsets of marginal zone B (MZB) cells differing in cell surface and transcriptomic profiles, clonal relationships to other subsets, enrichment of genes in the NOTCH pathway, distribution bias within splenic marginal zone microenvironment, and immunoglobulin repertoire diversity and hypermutation frequency. Each subset is present in spleen, gut-associated lymphoid tissue, mesenteric lymph nodes, and blood. MZB cells and the lineage from which they are derived are depleted in lupus nephritis. Here, we show that this depletion is of only one MZB subset. The other remains unchanged as a proportion of total B cells compared with health. Thus, it is important to factor MZB cell heterogeneity into studies of human B cell responses and pathology.

The Importance of Tissue Sanctuaries and Cellular Reservoirs of HIV-1.

There have been significant developments in the treatment of people living with HIV-1/AIDS with current antiretroviral therapies; however, these developments have not been able to achieve a functional or sterilizing cure for HIV-1. While there are multiple barriers, one such barrier is the existence of pharmacological sanctuaries and viral reservoirs where the concentration of antiretrovirals is suboptimal, which includes the gut-associated lymphoid tissue, central nervous system, lymph nodes, and myeloid cells. This review will focus on illustrating the significance of these sanctuaries, specific barriers to optimal antiretroviral concentrations in each of these sites, and potential strategies to overcome these barriers. Research and studies have shown that a uniform antiretroviral distribution is not achieved with current therapies. This may allow low-level replication associated with low antiretroviral concentrations in these sanctuaries/reservoirs. Many methods are being investigated to increase antiretroviral concentrations in these sites, such as blocking transporting enzymes functions, modulating transporter expression and nanoformulations of current antiretrovirals. While these methods have been shown to increase antiretroviral concentrations in the sanctuaries/reservoirs, no functional or sterilizing cure has been achieved due to these approaches. New methods of increasing antiretroviral concentrations at the specific sites of HIV-1 replication has the potential to target cellular reservoirs. In order to optimize antiretroviral distribution into viral sanctuaries/reservoirs, additional research is needed.

The porcine large intestine contains developmentally distinct submucosal lymphoid clusters and mucosal isolated lymphoid follicles

Gut-associated lymphoid tissues (GALT) serve as key priming sites for intestinal adaptive immune responses. Most of our understanding of GALT function and development arises from studies in mice. However, the diversity, structure and cellular composition of GALT differs markedly between mammalian species and the developmental window in which distinct GALT structures develop in large mammals remains poorly understood. Given the importance of pigs as models of human disease, as well as their role in livestock production, we adapted a recently developed protocol for the isolation of human GALT to assess the diversity, development and immune composition of large intestinal GALT in neonatal and adult pigs. We demonstrate that the large intestine of adult pigs contains two major GALT types; multifollicular submucosal GALT that we term submucosal lymphoid clusters (SLC) which develop prenatally, and as yet undescribed mucosal isolated lymphoid follicles (M-ILF), which arise after birth. Using confocal laser microscopy and flow cytometry, we additionally assess the microanatomy and lymphocyte composition of SLC and M-ILF, compare them to jejunal Peyer's patches (PP), and describe the maturation of these structures.Collectively, our results provide a deeper understanding of the diversity and development of GALT within the porcine large intestine.

Differential Modulation of Innate Antiviral Profiles in the Intestinal Lamina Propria Cells of Chickens Infected with Infectious Bursal Disease Viruses of Different Virulence.

Infectious bursal disease virus (IBDV) is one of the most important infectious diseases of poultry around the world. Gut-associated lymphoid tissues (GALT) are the first line of defense of the host against the infection. The purpose of this study was to investigate the role of innate immune antiviral signaling triggered by Toll-like receptor 3 (TLR3), as well as macrophage activation and cytokine response in the intestinal lamina propria (ILP) cells after the oral challenge of IBDV in relation to IBDV virulence and disease pathogenesis. The results showed that the expression levels of TLR3, IRF7, IFN-α/β and the corresponding downstream antiviral factors OAS, PKR and Mx were all upregulated in the SPF chicken ILP cells at 8 h post-infection (hpi) and 12 hpi. Similarly, macrophages were activated, with the initial macrophage M1 activation observed at 8 hpi, but then it rapidly shifted to a non-protective M2-type. Both Th1 (IFN-γ, TNF-α, IL-12) and Th2 (IL-4 and IL-10) types of cytokines were differentially upregulated during the early stage of infection; however, the Th1 cytokines exhibited stronger activation before 8 hpi compared to those of the Th2 cytokines. Interestingly, differential regulations of gene expression induced by different IBDV strains with different virulence were detected. The HLJ0504-like very virulent (vv) IBDV strain NN1172 induced stronger activation of TLR3-IFN-α/β pathway, macrophages and the Th1/2 cytokines’ expression, compared to those induced by the attenuated strain B87 at 8 hpi and 12 hpi in the ILP cells. In conclusion, the innate antiviral response mediated by the TLR3-IRF7 pathway, macrophage activation and cytokine expression in the GALT cells at the early stage of IBDV infection was differentially modulated, and the HLJ0504-like vvIBDV strain triggered stronger activation than the attenuated vaccine strain, and that may play an important role in the progression of disease.

Abstract P3-12-34: Antibiotic use and mortality from triple-negative breast cancer

Abstract Background: Gut-associated lymphoid tissue is the largest component of the body's immune system, as it influences both local and systemic immune responses. Gut microbiome dysbiosis related to antimicrobial exposure may be associated with decreased circulating and tumor-infiltrating lymphocytes and decreased immune repertoire, which may adversely impact survival in patients with triple-negative breast cancer (TNBC). We hypothesized that increasing antimicrobial exposure may lead to higher overall mortality (OM) and BC-specific mortality (BCM) in the presence of time-varying absolute lymphocyte count (ALC). Methods: Women with TNBC were identified in the Oncoshare database, a breast cancer registry that integrates data from the population-based SEER Registry and electronic medical records from two California healthcare systems (Stanford University and Sutter Health). We defined antibiotic use in three ways at each time point: (1) current use (“Antibiotic Use”), (2) total number of prescriptions (“Total Antibiotics”), and (3) total number of unique antibiotics prescribed (“Unique Antibiotics”) to study overall OM and BCM. We used marginal structural Cox proportional hazards multivariate models, with time-varying covariates (antibiotic use and ALC) to avoid immortal time bias. Results: 772 women were diagnosed with TNBC and treated from 2000-2014. The median overall follow-up time (including time to death) was 104 (IQR [61.66, 147.03]) months; the median follow-up time among those who were alive through the observation period was 121 (IQR [86.89, 160.93]) months. There were 654 (85%) patients who ever used antibiotics after TNBC diagnosis. There were 24/118 (20%) deaths among patients who never took antibiotics and 153/654 (23%) deaths in patients who ever took antibiotics during the study period. We previously showed that higher minimum ALC was associated with lower OM (HR 0.23, 95%CI (0.16, 0.35)) and BCM (HR 0.19, 95%CI (0.11, 0.34)) in TNBC patients from this cohort, with a significant association between higher peripheral lymphocyte count and tumor-infiltrating lymphocytes. Here, we found antibiotic use was associated with higher OM and BCM using definitions (2) and (3), but not definition (1). Model results are shown in the table and individual covariates are included for all definitions of antibiotic use. Conclusion: Higher number of antibiotic prescriptions and of unique antibiotics prescribed was associated with overall and breast cancer-specific mortality among women with TNBC. Future research on the role of the microbiome in mediating ALC and immune response may inform interventions to reduce TNBC mortality. Table. Overall and breast cancer-specific mortality among women with TNBC, Cox proportional hazards modelsOverall MortalityAntibiotic UseTotal AntibioticsUnique AntibioticsAntibiotic use1.54 (0.99, 2.39)Total number of antibiotics1.07* (1.04, 1.09)Number of unique antibiotics1.17* (1.12, 1.23)Age at diagnosis1.01* (1.00, 1.03)†1.01 (1.00, 1.02)1.01 (1.00, 1.03)Race/ethnicity (reference=White)Hispanic1.25 (0.70, 2.24)1.26 (0.70, 2.26)1.23 (0.69, 2.22)Non-Hispanic Asian Pacific Islander1.01 (0.64, 1.61)1.16 (0.74, 1.80)1.10 (0.69, 1.73)Non-Hispanic Black2.07* (1.12, 3.82)2.01* (1.10, 3.68)1.87* (1.03, 3.38)Neighborhood socioeconomic status quintile (reference=lowest)Second-lowest1.15 (0.49, 2.73)1.08 (0.47, 2.51)1.10 (0.49, 2.47)Middle1.22 (0.53, 2.81)1.05 (0.46, 2.40)1.06 (0.48, 2.35)Second-highest0.78 (0.34, 1.83)0.69 (0.30, 1.59)0.70 (0.31, 1.56)Highest0.96 (0.44, 2.11)0.82 (0.37, 1.78)0.80 (0.38, 1.70)Stage (reference=I)II1.72* (1.14, 2.59)1.61* (1.05, 2.45)1.71* (1.13, 2.57)III4.93* (3.08, 7.89)4.99* (3.10, 8.04)5.15* (3.20, 8.29)Grade (reference= 1)21.84 (0.66, 5.12)1.70 (0.61, 4.73)1.61 (0.57, 4.59)32.56 (0.95, 6.89)2.37 (0.88, 6.38)2.24 (0.82, 6.13)Received chemotherapy0.72 (0.45, 1.17)0.61 (0.37, 0.99)0.66 (0.41, 1.08)Received radiotherapy0.97 (0.70, 1.35)0.96 (0.69, 1.35)0.99 (0.71, 1.36)Used growth factors (e.g., filgrastim)1.38 (0.97, 1.97)1.31 (0.90, 1.93)1.15 (0.79, 1.67)† 95% CI lower bound is rounded from 1.001. * p < 0.05 Breast Cancer-Specific MortalityAntibiotic UseTotal AntibioticsUnique AntibioticsAntibiotic use1.50 (0.90, 2.47)Total number of antibiotics1.07* (1.04, 1.10)Number of unique antibiotics1.18* (1.12, 1.25)Age at diagnosis1.00 (0.98, 1.01)1.00 (0.98, 1.01)1.00 (0.98, 1.01)Race/ethnicity (reference=White)Hispanic1.36 (0.71, 2.61)1.36 (0.71, 2.60)1.34 (0.69, 2.58)Non-Hispanic Asian Pacific Islander1.41 (0.86, 2.31)1.48 (0.91, 2.40)1.47 (0.90, 2.40)Non-Hispanic Black1.69 (0.76, 3.76)1.66 (0.76, 3.60)1.55 (0.70, 3.43)Neighborhood socioeconomic status quintile (reference=lowest)Second-lowest1.54 (0.52, 4.52)1.71 (0.57, 5.17)1.59 (0.53, 4.79)Middle1.50 (0.53, 4.26)1.61 (0.55, 4.71)1.48 (0.51, 4.31)Second-highest1.15 (0.41, 3.27)1.17 (0.40, 3.41)1.12 (0.39, 3.26)Highest1.14 (0.42, 3.06)1.19 (0.43, 3.30)1.05 (0.38, 2.92)Stage (reference=I)II2.39* (1.37, 4.17)2.43* (1.39, 4.27)2.45* (1.40, 4.28)III7.96* (4.31, 14.70)8.35* (4.49, 15.53)8.48* (4.55, 15.80)Grade (reference= 1)21.12 (0.37, 3.36)1.01 (0.33, 3.05)0.91 (0.30, 2.81)31.67 (0.60, 4.68)1.52 (0.54, 4.26)1.42 (0.50, 4.04)Received chemotherapy0.64 (0.36, 1.14)0.61 (0.34, 1.10)0.60 (0.33, 1.09)Received radiotherapy0.95 (0.64, 1.39)0.91 (0.62, 1.33)0.95 (0.65, 1.38)Used growth factors (e.g., filgrastim)1.05 (0.70, 1.57)0.94 (0.63, 1.41)0.88 (0.58, 1.33)NOTE: * denotes p<0.05 Citation Format: Julia D Ransohoff, Karen Andrade, Natasha Purington, Vidhya Balasubramanian, Summer Han, Mina Liu, Jennifer Caswell-Jin, George Sledge, Ami Bhatt, Allison Kurian. Antibiotic use and mortality from triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-12-34.

Ex Vivo Differential Responsiveness to Clostridium perfringens and Lactococcus lactis by Avian Small Intestine Macrophages and T Cells.

Tissue resident immune system cells in the chicken intestine play a significant role in the protection against pathogens. However, very little is known about these cells. The current study was conducted to further characterize chicken intestinal immune system cells. Furthermore, this study aimed to assess the immune modulatory action of a highly virulent Clostridium perfringens, a commonly found chicken intestinal microbe, in comparison with a non-commensal, Lactococcus lactis, on intestine-derived immune system cells. The results demonstrated varying distribution of innate and adaptive immune cells along the avian gut-associated lymphoid tissue (GALT) in the duodenum, jejunum, ileum, and cecal tonsils. In addition, steady-state and tissue-specific presence of CD25+ cells among αβ and γδ T-cell subsets was assessed along the intestine. Ex vivo stimulation with C. perfringens or L. lactis resulted in a significant increase in the frequency of CD25+ T cells (γδ and αβ T cells). In addition, significantly more cell death was observed in ex vivo stimulation with C. perfringens, which was indirectly correlated with a decrease in macrophage activation based on nitric oxide (NO) production with no effect on lymphoid cell responsiveness as per intracellular interferon (IFN)-gamma (γ) staining. Ex vivo stimulation with L. lactis activated γδ T cells and αβ T cells, based on intracellular IFN-γ staining, while it had limited effect on macrophages. However, the ability of γδ and αβ T cells to produce IFN-γ and the ability of macrophages production of NO was rescued in the presence of L. lactis. These results demonstrate the potential application of L. lactis, as a probiotic, against virulent C. perfringens infection in chicken.

Lipoteichoic Acid from Lacticaseibacillus rhamnosus GG Modulates Dendritic Cells and T Cells in the Gut

Lipoteichoic acid (LTA) from Gram-positive bacteria exerts different immune effects depending on the bacterial source from which it is isolated. Lacticaseibacillus rhamnosus GG LTA (LGG-LTA) oral administration reduces UVB-induced immunosuppression and skin tumor development in mice. In the present work, we evaluate the immunomodulatory effect exerted by LGG-LTA in dendritic cells (DC) and T cells, both in vitro and in the gut-associated lymphoid tissue (GALT). During cell culture, LTA-stimulated BMDC increased CD86 and MHC-II expression and secreted low levels of pro and anti-inflammatory cytokines. Moreover, LTA-treated BMDC increased T cell priming capacity, promoting the secretion of IL-17A. On the other hand, in orally LTA-treated mice, a decrease in mature DC (lamina propria and Peyer’s patches) was observed. Concomitantly, an increase in IL-12p35 and IFN-γ transcription was presented (lamina propria and Peyer’s Patches). Finally, an increase in the number of CD103+ DC was observed in Peyer’s patches. Together, our data demonstrate that LGG-LTA activates DC and T cells. Moreover, we show that a Th1-biased immune response is triggered in vivo after oral LTA administration. These effects justify the oral LTA activity previously observed.

The Role of the Lymphatic System in the Pathogenesis and Treatment of Inflammatory Bowel Disease.

Although the number of therapeutic options for the treatment of inflammatory bowel disease (IBD) has increased in recent years, patients suffer from decreased quality of life due to non-response or loss of response to the currently available treatments. An increased understanding of the disease’s etiology could provide novel insights for treatment strategies in IBD. Lymphatic system components are generally linked to immune responses and presumably related to inflammatory diseases pathophysiology. This review aims to summarize findings on immune-mediated mechanisms in lymphoid tissues linked with IBD pathogenesis and (potential) novel treatments. Enhanced innate and adaptive immune responses were observed in mesenteric lymph nodes (MLNs) and other lymphoid structures, such as Peyer’s patches, in patients with IBD and in animal models. Furthermore, the phenomenon of lymphatic obstruction in the form of granulomas in MLNs and lymphatic vessels correlates with disease activity. There is also evidence that abnormalities in the lymphatic stromal components and lymph node microbiome are common in IBD and could be exploited therapeutically. Finally, novel agents targeting lymphocyte trafficking have been added to the treatment armamentarium in the field of IBD. Overall, gut-associated lymphoid tissue plays a key role in IBD immunopathogenesis, which could offer novel therapeutic targets.