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Vitamin A (VA) deficiency (VAD) is observed in both humans and mice with lupus nephritis. However, whether VAD is a driving factor for accelerated progression of lupus nephritis is unclear. In this study, we investigated the effect of VAD on the progression of lupus nephritis in a lupus-prone mouse model, MRL/lpr. We initiated VAD either during gestation or after weaning to reveal a potential time-dependent effect. We found exacerbated lupus nephritis at ∼15 wk of age with both types of VAD that provoked tubulointerstitial nephritis leading to renal failure. This was concomitant with significantly higher mortality in all VAD mice. Importantly, restoration of VA levels after weaning reversed VAD-induced mortality. These results suggest VAD-driven acceleration of tubulointerstitial lupus nephritis. Mechanistically, at the earlier time point of 7 wk of age and before the onset of clinical lupus nephritis, continued VAD (from gestation until postweaning) enhanced plasma cell activation and augmented their autoantibody production, while also increasing the expansion of T lymphocytes that could promote plasma cell autoreactivity. Moreover, continued VAD increased the renal infiltration of plasmacytoid dendritic cells. VAD initiated after weaning, in contrast, showed modest effects on autoantibodies and renal plasmacytoid dendritic cells that were not statistically significant. Remarkably, analysis of gene expression in human kidney revealed that the retinoic acid pathway was decreased in the tubulointerstitial region of lupus nephritis, supporting our findings in MRL/lpr mice. Future studies will elucidate the underlying mechanisms of how VAD modulates cellular functions to exacerbate tubulointerstitial lupus nephritis.

Commensal bacteria and the immune system have a close and strong relationship that maintains a balance to control inflammation. Alterations of the microbiota, known as dysbiosis, can direct reactivity to self-antigens not only in the intestinal mucosa but also at the systemic level. Our laboratory previously reported gut dysbiosis, particularly lower abundance of bacteria in the familyLactobacillaceae, in lupus-prone MRL/lprmice, a model of systemic autoimmunity. Restoring the microbiota with a mix of 5 differentLactobacillusspecies (spp.),L. reuteri, L. oris, L. johnsonii, L. gasseriandL. rhamnosus, attenuated lupus-liked clinical signs, including splenomegaly and lymphadenopathy. However, our understanding of the mechanism was limited. In this study, we first investigated the effects of individual species. Surprisingly, none of the species individually recapitulated the benefits of the mix. Instead,Lactobacillusspp. acted synergistically to attenuate splenomegaly and renal lymphadenopathy through secreted factors and a CX3CR1-dependent mechanism. Interestingly, oral administration of MRS broth exerted the same benefits likely through increasing the relative abundance of endogenousLactobacillusspp. Mechanistically, we found increased percentages of FOXP3-negative type 1 regulatory T cells with administration of the mix in both spleen and mesenteric lymph nodes. In addition, oral gavage ofLactobacillusspp. decreased the percentage of central memory T cells while increasing that of effector memory T cells in the lymphoid organs. Furthermore, a decreased percentage of double negative T cells was observed in the spleen with the mix. These results suggest thatLactobacillusspp. might act on T cells to attenuate splenomegaly and lymphadenopathy. Together, this study advances our understanding of howLactobacillusspp. attenuate lupus in MRL/lprmice. The synergistic action of these bacteria suggests that multiple probiotic bacteria in combination may dampen systemic autoimmunity and benefit lupus patients.

Lactobacillus rhamnosus GG is a widely used probiotic to improve host intestinal health, promote growth, reduce diarrhea, and modulate immunity. In recent years, the bacterial quorum sensing system has attracted much attention; however, there has not been much research on the effect of the LuxS/AI-2 quorum sensing system of Lactobacillus on bacteriostasis, microbial ecology balance, and immune regulation in intestine.

Systemic lupus erythematosus (SLE) is an autoimmune disorder with no known cure and is characterized by persistent inflammation in many organs, including the kidneys. Under such circumstances, the kidney loses its ability to clean waste from the blood and regulate salt and fluid concentrations, eventually leading to renal failure. Women, particularly those of childbearing age, are diagnosed nine times more often than men. Kidney disease is the leading cause of mortality in SLE patients. The present protocol describes a quick and simple method to measure excreted protein levels in collected urine, tracking lupus progression over time. In addition, an approach to isolate kidney mononuclear cells is provided based on size and density selection to investigate renal infiltration of leukocytes. Furthermore, an immunohistochemical method has been developed to characterize protein deposition in the glomeruli and leukocyte infiltration in the tubulointerstitial space. Together, these methods can help investigate the progression of chronic inflammation associated with the kidneys of lupus-prone MRL/lpr mice.

With the rising prevalence of autoimmune diseases, the role of the environment, specifically the gut microbiota, in disease development has grown to be a major area of study. Recent advances show a relationship and possible cause and effect between the gut microbiota and the initiation or exacerbation of autoimmune diseases. Furthermore, microbial dysbiosis and leaky gut are frequent phenomena in both human autoimmune diseases and the murine autoimmunity models. This review will focus on literature in recent years concerning the gut microbiota and leaky gut in relation to the autoimmune diseases, including systemic lupus erythematosus, type 1 diabetes, and multiple sclerosis. Published version

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that are not only driven by genetic factors, but also environmental factors that can trigger the onset and/or worsen the disease course. A previously published study on the dynamics of the gut microbiota in lupus-prone MRL/lpr female mice showed how changes of the gut microbiota can alter disease progression. Here, a protocol is described for extracting representative samples from the gut microbiota for studies of autoimmunity. Microbiota samples are collected from the anus and processed, from which the DNA is extracted using a phenol-chloroform method and purified by alcohol precipitation. After PCR is performed, purified amplicons are sequenced using a Next Generation Sequencing platform at Argonne National Laboratory. Finally, the 16S ribosomal RNA gene sequencing data is analyzed. As an example, data obtained from gut microbiota comparisons of MRL/lpr mice with or without CX3CR1 are shown. Results showed significant differences in genera containing pathogenic bacteria such as those in the phylum Proteobacteria, as well as the genus Bifidobacterium, which is considered part of the healthy commensal microbiota. In summary, this simple, cost-effective DNA isolation method is reliable and can help the investigation of gut microbiota changes associated with autoimmune diseases.

Synovial inflammation is a central feature of osteoarthritis (OA), elicited when local regulatory macrophages (M2-like) become overwhelmed, activating an inflammatory response (M1-like). Bone marrow mononuclear cells (BMNC) are a source of naive macrophages capable of reducing joint inflammation and producing molecules essential for cartilage metabolism. This study investigated the response of BMNC to normal (SF) and inflamed synovial fluid (ISF). Equine BMNC cultured in autologous SF or ISF (n = 8 horses) developed into macrophage-rich cultures with phenotypes similar to cells native to normal SF and became more confluent in ISF (~100%) than SF (~25%). BMNC cultured in SF or ISF were neither M1- nor M2-like, but exhibited aspects of both phenotypes and a regulatory immune response, characterized by increasing counts of IL-10+ macrophages, decreasing IL-1β concentrations and progressively increasing IL-10 and IGF-1 concentrations. Changes were more marked in ISF and suggest that homeostatic mechanisms were preserved over time and were potentially favored by progressive cell proliferation. Collectively, our data suggest that intra-articular BMNC could increase synovial macrophage counts, potentiating the macrophage- and IL-10-associated mechanisms of joint homeostasis lost during the progression of OA, preserving the production of cytokines involved in tissue repair (PGE2 , IL-10) generally impaired by frequently used corticosteroids.

The immune system is an efficiently toned machinery that discriminates between friends and foes for achieving both host defense and homeostasis. Deviation of immune recognition from foreign to self and/or long-lasting inflammatory responses results in the breakdown of tolerance. Meanwhile, educating the immune system and developing immunological memory are crucial for mounting defensive immune responses while protecting against autoimmunity. Still to elucidate is how diverse environmental factors could shape autoimmunity. The emergence of a world pandemic such as SARS-CoV-2 (COVID-19) not only threatens the more vulnerable individuals including those with autoimmune conditions but also promotes an unprecedented shift in people’s dietary approaches while urging for extraordinary hygiene measures that likely contribute to the development or exacerbation of autoimmunity. Thus, there is an urgent need to understand how environmental factors modulate systemic autoimmunity to better mitigate the incidence and or severity of COVID-19 among the more vulnerable populations. Here, we discuss the effects of diet (macronutrients and micronutrients) and hygiene (the use of disinfectants) on autoimmunity with a focus on systemic lupus erythematosus.

MRL/lpr mice have been extensively used as a murine model of lupus. Disease progression in MRL/lpr mice can differ among animal facilities, suggesting a role for environmental factors.We noted a phenotypic drift of our in-house colony, which was the progeny of mice obtained from The Jackson Laboratory (JAX; stocking number 000485), that involved attenuated glomerulonephritis, increased splenomegaly, and reduced lymphadenopathy. To validate our in-house mice as a model of lupus, we compared these mice with those newly obtained from JAX, which were confirmed to be genetically identical to our in-house mice. Surprisingly, the new JAX mice exhibited a similar phenotypic drift, most notably the attenuation of glomerulonephritis. Interestingly, our in-house colony differed from JAX mice in body weight and kidney size (both sexes), as well as in splenic size, germinal center formation, and level of anti-dsDNA auto-IgG in the circulation (male only). In addition, we noted differential expression of microRNA (miR)-21 and miR-183 that might explain the splenic differences in males. Furthermore, the composition of gut microbiota was different between in-house and new JAX mice at early time points, which might explain some of the renal differences (e.g., kidney size). However, we could not identify the reason for attenuated glomerulonephritis, a shared phenotypic drift between the two colonies. It is likely that this was due to certain changes of environmental factors present in both JAX and our facilities. Taken together, these results suggest a significant phenotypic drift in MRL/lpr mice in both colonies that may require strain recovery from cryopreservation. This work was supported by internal funding from Virginia-Maryland College of Veterinary Medicine and National Institutes of Health Grants AR067418 and AR073240 (X.M.L.). Published version

Background: Intermittent fasting (IF) can reduce energy intake and body weight (BW). Melatonin has many known functions, which include reducing appetite and preventing excessive weight gain.Objective: This study aimed to investigate the effects of IF on body fat and the gut microbiota and metabolome as well as a potential interaction with melatonin.Methods: Male C57BL/6J mice (23.0 ± 0.9 g, 6 wk old) were randomly assigned into four groups (12 mice/group): control (C), intermittent fasting (F), melatonin (M), and intermittent fasting plus melatonin (MF). The C and M groups mice were provided with ad libitum access to food and water, while the F and MF groups underwent alternative-day feed deprivation (15 cycles total). Melatonin was administered in the drinking water of the M and MF groups. Blood, epididymal fat, liver tissue, and intestinal tissue and contents were collected for lab measurements, histology, and microbiota and metabolome analysis. Main effects and interactions were tested by 2-factor ANOVA.Results: IF significantly reduced BW gain and serum glucose, total cholesterol (TC) and triglyceride (TG) levels. Adipocyte size significantly decreased with IF, then the number of adipocytes per square millimeter significantly increased (P < 0.05). Compared to the C group, the M and MF groups had significantly higher serum melatonin levels (17 and 21%, respectively), although melatonin monotherapy had no effect on serum parameters and adipocytes. There was no interaction between IF and melatonin on BW gain and serum parameters except for on adipocyte area and number per square millimeter, Bacteroidetes and Akkermansia bacterial abundance, and the levels of the intestinal metabolites alanine, valine and isoleucine. IF changed the intestinal microbiota structure, with the F and MF groups clearly separating from the C and M groups. Metabolomic analysis showed that there was obvious separation between all four groups.Conclusions: IF, but neither melatonin nor the interaction between IF and melatonin, could alter intestinal microbiota and metabolism and prevent obesity by reducing BW gain, serum glucose, TC, and TG, and adipocyte size in mice.

BACKGROUND Lactobacillus rhamnosus GG culture supernatant (LGGs) promotes intestinal integrity and ameliorates acute liver injury induced by alcohol in mice. OBJECTIVES The aim of this study was to investigate the protective effects and molecular mechanisms of Lactobacillus reuteri ZJ617 culture supernatant (ZJ617s) on acute liver injury induced by lipopolysaccharide (LPS) in mice. METHODS Male C57BL/6 mice (20 ± 2 g, 8 wk old) were randomly divided into 4 groups (6 mice/group): oral inoculation with phosphate-buffered saline (control), intraperitoneal injection of LPS (10 mg/kg body weight) (LPS), oral inoculation with ZJ617s 2 wk before intraperitoneal injection of LPS (ZJ617s + LPS), or oral inoculation with LGGs 2 wk before intraperitoneal injection of LPS (LGGs + LPS). Systemic inflammation, intestinal integrity, biomarkers of hepatic function, autophagy, and apoptosis signals in the liver were determined. RESULTS Twenty-four hours after LPS injection, the activities of serum alanine transaminase and aspartate transaminase were 32.2% and 30.3% lower in the ZJ617s + LPS group compared with the LPS group, respectively (P

Osteoarthritis (OA) is characterized by macrophage-driven synovitis. Macrophages promote synovial health but become inflammatory when their regulatory functions are overwhelmed. Bone marrow mononuc...

Significance Infants are born without an established gut microbiota, which develops rapidly after birth and is shaped by the maternal microbiota. However, how the maternal microbiota, through shaping the neonatal microbiota, would affect the establishment of a strong immune system in neonates remains unclear. Here, we show mechanistically how the maternal microbiota regulates the de novo production of neonatal IgA., Infants are prone to enteric infections due to an underdeveloped immune system. The maternal microbiota, through shaping the neonatal microbiota, helps establish a strong immune system in infants. We and others have observed the phenomenon of enhanced early neonatal immunoglobulin A (IgA) production in preweaning immunocompetent mice nursed by immunodeficient dams. Here, we show that this enhancement of IgA in neonates results from maternally derived microbiota. In addition, we have found that the neonatal IgA production can be induced by Lactobacillus reuteri, which is enriched in the milk of immunodeficient dams. Moreover, we show that while the production of neonatal IgA is dependent on neonatal T cells, the immunodeficient maternal microbiota-mediated enhancement of neonatal IgA has a T cell–independent component. Indeed, this enhancement may be dependent on type 3 innate lymphoid cells in the neonatal small intestinal lamina propria. Interestingly, maternal microbiota-induced neonatal IgA does not cross-react with common enteric pathogens. Future investigations will determine the functional consequences of having this extra IgA.

The ability to adhere to the intestinal mucus layer is an important property of probiotic bacteria. Lactobacillus reuteri strains ZJ615 and ZJ617 show low and high adhesion, respectively, to intestinal epithelial cells. In this study, we quantified bacterial cell wall-associated glyceraldehyde-3-phosphate dehydrogenases (cw-GAPDH) and bacterial cell membrane permeability in both strains using immunoblotting and flow cytometry, respectively. Highly adhesive L. reuteri ZJ617 possessed significantly more cw-GAPDH, higher cell membrane permeability, and significantly higher adhesive ability toward mucin compared with low-adhesive L. reuteri ZJ615. In vitro adhesion studies and analysis of interaction kinetics using the Octet, the system revealed significantly decreased interaction between L. reuteri and mucin when mucin was oxidized when bacterial surface proteins were removed when bacteria were heat-inactivated at 80 &deg, C for 30 min, and when the interaction was blocked with an anti-GAPDH antibody. SWISS-MODEL analysis suggested intensive interactions between mucin glycans (GalNAc&alpha, 1-O-Ser, GalNAc&alpha, Ser, and Gal&beta, 3GalNAc) and GAPDH. Furthermore, in vivo studies revealed significantly higher numbers of bacteria adhering to the jejunum, ileum, and colon of piglets orally inoculated with L. reuteri ZJ617 compared with those inoculated with L. reuteri ZJ615, this led to a significantly decreased rate of diarrhea in piglets inoculated with L. reuteri ZJ617. In conclusion, there are strong correlations among the abundance of cw-GAPDH in L. reuteri, the ability of the bacterium to adhere to the host, and the health benefits of this probiotic.

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease. The cause of SLE is not only genetic; in fact, environmental factors may play a more important role in disease development. The MRL/lpr mouse model lupus-like symptoms due to multiple SLE susceptible loci in the MRL background, and offers an accelerated model compared to the MRL parent strain due to the Faslpr mutation. Recently, our laboratory witnessed a loss of disease phenotype in our in-house colony of MRL/lpr mice. We thus compared mice newly obtained from The Jackson Laboratory (JAX; Stock Number 000485) to our in-house mice. The single-nucleotide polymorphism (SNP) analysis showed no genetic drift, suggesting that environmental factors could be triggering a phenotypic drift. Surprisingly, the newly purchased JAX mice also had attenuation of glomerulonephritis. Even though JAX mice manifested similar attenuation of lupus nephritis, our in-house colony showed differences in organ weights. Furthermore, males showed a significantly higher level of anti-double stranded DNA auto-IgG consistent with germinal center maturation. In addition, in-house males had significantly higher levels of microRNA-21 and microRNA-183 explaining spleen size difference. Moreover, the composition of gut microbiota was different between in-house and new JAX mice at early age, with many groups of bacteria differing at later time points, which might explain some of the phenotypic differences. These results suggest that microRNAs and gut microbiota might be responsible for the phenotypic differences of MRL/lpr mice in JAX and our colonies as they were genetically identical. On the other hand, the attenuation of nephritis in both groups requires further investigation. Supported by R01AR073240

The ability to adhere to the intestinal mucus layer is an important property of probiotic bacteria. Lactobacillus reuteri strains ZJ615 and ZJ617 show low and high adhesion, respectively, to intestinal epithelial cells. In this study, we quantified bacterial cell wall-associated glyceraldehyde-3-phosphate dehydrogenases (cw-GAPDH) and bacterial cell membrane permeability in both strains using immunoblotting and flow cytometry, respectively. Highly adhesive L. reuteri ZJ617 possessed significantly more cw-GAPDH, higher cell membrane permeability, and significantly higher adhesive ability toward mucin compared with low-adhesive L. reuteri ZJ615. In vitro adhesion studies and analysis of interaction kinetics using the Octet, the system revealed significantly decreased interaction between L. reuteri and mucin when mucin was oxidized when bacterial surface proteins were removed when bacteria were heat-inactivated at 80 °C for 30 min, and when the interaction was blocked with an anti-GAPDH antibody. SWISS-MODEL analysis suggested intensive interactions between mucin glycans (GalNAcα1-O-Ser, GalNAcαSer, and Galβ3GalNAc) and GAPDH. Furthermore, in vivo studies revealed significantly higher numbers of bacteria adhering to the jejunum, ileum, and colon of piglets orally inoculated with L. reuteri ZJ617 compared with those inoculated with L. reuteri ZJ615; this led to a significantly decreased rate of diarrhea in piglets inoculated with L. reuteri ZJ617. In conclusion, there are strong correlations among the abundance of cw-GAPDH in L. reuteri, the ability of the bacterium to adhere to the host, and the health benefits of this probiotic.

Autoimmune diseases, such as systemic lupus erythematosus, are characterized by excessive inflammation in response to self-antigens. Loss of appropriate immunoregulatory mechanisms contribute to disease exacerbation. We previously showed the suppressive effect of vancomycin treatment during the "active-disease" stage of lupus. In this study, we sought to understand the effect of the same treatment given before disease onset. To develop a model in which to test the regulatory role of the gut microbiota in modifying autoimmunity, we treated lupus-prone mice with vancomycin in the period before disease development (3-8 weeks of age). We found that administration of vancomycin to female MRL/lpr mice early, only during the pre-disease period but not from 3 to 15 weeks of age, led to disease exacerbation. Early vancomycin administration also reduced splenic regulatory B (Breg) cell numbers, as well as reduced circulating IL-10 and IL-35 in 8-week old mice. Further, we found that during the pre-disease period, administration of activated IL-10 producing Breg cells to mice treated with vancomycin suppressed lupus initiation, and that bacterial DNA from the gut microbiota was an inducer of Breg function. Oral gavage of bacterial DNA to mice treated with vancomycin increased Breg cells in the spleen and mesenteric lymph node at 8 weeks of age and reduced autoimmune disease severity at 15 weeks. This work suggests that a form of oral tolerance induced by bacterial DNA-mediated expansion of Breg cells suppress disease onset in the autoimmune-prone MRL/lpr mouse model. Future studies are warranted to further define the mechanism behind bacterial DNA promoting Breg cells. NIAMSUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Arthritis & Musculoskeletal & Skin Diseases (NIAMS) [AR073240, AR067418] NIAMS AR073240, AR067418 (XL).

Background: The olive extract contains compounds with antioxidant and anti-inflammatory properties. This study was designed to investigate whether olive cake extract, enriched with maslinic acid and hydroxytyrosol, alleviates the lipopolysaccharides (LPS)-induced oxidative stress, inflammation and intestinal villus damage in piglets.Methods: Thirty weaned piglets (6.9±0.9 kg) were assigned to five groups using a randomized complete block design. Piglets were fed a basal diet before intraperitoneal (i.p.) injection of physiological saline (C); fed a basal diet alone (CL) or fed a basal diet plus olive extract (OL), antibiotics (AL), or olive extract and antibiotics (OAL) before i.p. injection of LPS. The feeding lasted for 2 weeks. Piglets were euthanized 4h after LPS injection. Systemic anti-oxidant and inflammation levels were measured and villus morphology in the intestine was examined.Results: Compared with those in the C group, piglets in the CL group had significantly lower GSH-Px, SOD, ALB levels and higher MDA, NO, LDH, ALT and AST levels in the serum (PPPPPPLactobacillus and Clostridium at genus level.Conclusion: Dietary supplementation with olive extract maslinic acid and hydroxytyrosol improved anti-oxidative and anti-inflammatory capacity, intestinal structure morphology, and increased the abundance of beneficial intestinal bacteria in weaned piglets challenged by LPS.

The intestine is a particularly dynamic environment in which the host constantly interacts with trillions of symbiotic bacteria called the microbiota. Using quorum sensing (QS) communication, bacteria can coordinate their social behavior and influence host cell activities in a non-invasive manner. Nowadays, a large amount of research has greatly spurred the understanding of how bacterial QS communication regulates bacterial cooperative behaviors due to coexistence and host-microbe interactions. In this review, we discuss bacterial QS in the gut and its role in biofilm formation. As a biological barrier, the mucosal immune system can effectively prevent pathogenic microorganisms and other immunogenic components from entering the internal environment of the host. We focus on the relationship between biofilm and intestinal mucosal immunity, and how probiotic bacteria may regulate them. This review is to provide a theoretical basis for the development of new techniques including probiotics targeting the intestinal barrier function, thereby improving gut health.

We previously showed that all-trans-retinoic acid (tRA), an active metabolite of vitamin A, exacerbated pre-existing autoimmunity in lupus; however, its effects before the development of autoimmunity are unknown. Here, using a pristane-induced model, we show that tRA exerts differential effects when given at the initiation vs. continuation phase of lupus. Unlike tRA treatment during active disease, pre-pristane treatment with tRA aggravated glomerulonephritis through increasing renal expression of pro-fibrotic protein laminin β1, activating bone marrow conventional dendritic cells (cDCs), and upregulating the interaction of ICAM-1 and LFA-1 in the spleen, indicating an active process of leukocyte activation and trafficking. Transcriptomic analysis revealed that prior to lupus induction, tRA significantly upregulated the expression of genes associated with cDC activation and migration. Post-pristane tRA treatment, on the other hand, did not significantly alter the severity of glomerulonephritis; rather, it exerted immunosuppressive functions of decreasing circulatory and renal deposition of autoantibodies as well as suppressing the renal expression of proinflammatory cytokines and chemokines. Together, these findings suggest that tRA differentially modulate lupus-associated kidney inflammation depending on the time of administration. Interestingly, both pre- and post-pristane treatments with tRA reversed pristane-induced leaky gut and modulated the gut microbiota in a similar fashion, suggesting a gut microbiota-independent mechanism by which tRA affects the initiation vs. continuation phase of lupus.

BACKGROUND Probiotic Lactobacillius rhamnosus GG (LGG) shows beneficial immunomodulation on cultured cell lines in vitro and in mouse models. OBJECTIVE The aim was to investigate the effects of LGG on intestinal injury and the underlying mechanisms by elucidating inflammatory signaling pathways and metabolomic response to LPS stimulation in the piglet intestine. METHODS Piglets (Duroc × Landrace × Large White, including males and female; 8.6 ± 1.1 kg) aged 28 d were assigned to 3 groups (n = 6/group): oral inoculation with PBS for 2 wk before intraperitoneal injection of physiological saline [control (CON)] or LPS (25 μg/kg body weight; LPS) or oral inoculation with LGG for 2 wk before intraperitoneal injection of LPS (LGG+LPS). Piglets were killed 4 h after LPS injection. Systemic inflammation, intestinal integrity, inflammation signals, and metabolomic characteristics in the intestine were determined. RESULTS Compared with CON, LPS stimulation significantly decreased ileal zonula occludens 1 (ZO-1; 44%), claudin-3 (44%), and occludin (41%) expression; increased serum diamineoxidase (73%), D-xylose (19%), TNF-α (43%), and IL-6 (55%) concentrations; induced p38 mitogen-activated protein kinase (p38 MAPK; 85%), extracellular signal-regulated kinase (ERK; 96%), and NF-κB p65 phosphorylation (37%) (P

The aim of this study was to determine whether Lactobacillus rhamnosus GG (LGG) components (surface layer protein, SLP; genomic DNA, gDNA; unmethylated cytosine-phosphate-guanine-containing oligodeoxynucleotide, CpG-ODN), alone or in combination, could affect immunomodulation, and evaluate the signalling mechanism in mouse macrophage RAW264.7 cells challenged with lipopolysaccharide (LPS). LGG components were used to treat cells before LPS stimulation. Cytokine and Toll-like receptor (TLR) expression were assessed using real-time quantitative PCR (RT-qPCR). Mitogen-activated protein kinase (MAPK), extracellular regulated protein kinase (ERK) and nuclear factor-kappa B (NF-κB) signalling pathways were evaluated using immunoblots and immunofluorescence. SLP or SLP + gDNA pre-treatment significantly reduced the LPS-induced mRNA expression of tumour necrosis factor alpha (TNF-α). Pre-treatment with LGG single components (SLP, gDNA or CpG) or their combinations (SLP + gDNA or SLP + CpG) significantly decreased the LPS-induced interleukin-6 (IL-6) mRNA level (P 0·05). Pre-treatment with SLP or gDNA, alone or in combination, significantly suppressed LPS-induced TLR2 and TLR4 mRNA levels (P 0·05). SLP pre-treatment also significantly decreased the LPS-induced expression of TLR9 (P 0·05). Pre-treatment with LGG single components or combinations significantly suppressed the LPS-induced phosphorylation levels of ERK (P 0·05). In conclusion, pre-incubation with LGG components, singly or in combination, generally inhibited the activation of TLR, MAPK and NF-κB signalling pathways in LPS-stimulated cells, leading to attenuated inflammatory cytokine TNF-α and IL-6 production. These results indicate that nonviable probiotic LGG components exert an anti-inflammation effect on epithelial cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus rhamnosus GG (LGG) is widely used as probiotics. However, its main components are not well known for affecting immunomodulation. This study investigated the effects of pre-treatments with different components such as surface layer protein, genomic DNA and unmethylated cytosine-phosphate-guanine-containing oligodeoxynucleotides, alone or in combination on immunomodulation, and evaluated the signalling mechanism in mouse macrophage RAW264.7 cells challenged with lipopolysaccharide. Pre-incubation with components alone or in combination generally inhibited the activation of Toll-like receptor, mitogen-activated protein kinases, extracellular regulated protein kinases and nuclear factor-kappa B signalling pathways in lipopolysaccharide-stimulated cells, which generally leads to attenuated inflammatory cytokine interleukin-6 and tumour necrosis factor alpha production. These results indicate that nonviable probiotic LGG components exert an anti-inflammation effect on epithelial cells.

Background Dysbiosis of gut microbiota exists in the pathogenesis of many autoimmune diseases, including systemic lupus erythematosus (lupus). Lupus patients who experienced pregnancy usually had more severe disease flares post-delivery. However, the possible role of gut microbiota in the link between pregnancy and exacerbation of lupus remains to be explored. Results In the classical lupus mouse model MRL/lpr, we compared the structures of gut microbiota in pregnant and lactating individuals vs. age-matched naïve mice. Consistent with studies on non-lupus mice, both pregnancy and lactation significantly changed the composition and diversity of gut microbiota. Strikingly, modulation of gut microbiota using the same strategy resulted in different disease outcomes in postpartum (abbreviated as “PP,” meaning that the mice had undergone pregnancy and lactation) vs. control (naïve; i.e., without pregnancy or lactation) MRL/lpr females; while vancomycin treatment attenuated lupus in naïve mice, it did not do so, or even exacerbated lupus, in PP mice. Lactobacillus animalis flourished in the gut upon vancomycin treatment, and direct administration of L. animalis via oral gavage recapitulated the differential effects of vancomycin in PP vs. control mice. An enzyme called indoleamine 2,3-dioxygenase was significantly inhibited by L. animalis; however, this inhibition was only apparent in PP mice, which explained, at least partially, the lack of beneficial response to vancomycin in these mice. The differential production of immunosuppressive IL-10 and proinflammatory IFNγ in PP vs. control mice further explained why the disease phenotypes varied between the two types of mice bearing the same gut microbiota remodeling strategy. Conclusions These results suggest that pregnancy and lactation interfere with the response of autoimmunity to modulation of gut microbiota. Further studies are necessary to better understand the complex relationship between pregnancy and lupus. Electronic supplementary material The online version of this article (10.1186/s40168-019-0720-8) contains supplementary material, which is available to authorized users.

Probiotics are being investigated for the treatment of autoimmune disease by re-balancing dysbiosis induced changes in the immune system. Pregnancy is a health concern surrounding autoimmune disease, both for the mother and her child. Probiotics for maternity are emerging on the market and have gained significant momentum in the literature. Thus far, evidence supports that probiotics alter the structure of the normal microbiota and the microbiota changes significantly during pregnancy. The interaction between probiotics-induced changes and normal changes during pregnancy is poorly understood. Furthermore, there is emerging evidence that the maternal gut microbiota influences the microbiota of offspring, leading to questions on how maternal probiotics may influence the health of neonates. Underpinning the development and balance of the immune system, the microbiota, especially that of the gut, is significantly important, and dysbiosis is an agent of immune dysregulation and autoimmunity. However, few studies exist on the implications of maternal probiotics for the outcome of pregnancy in autoimmune disease. Is it helpful or harmful for mother with autoimmune disease to take probiotics, and would this be protective or pathogenic for her child? Controversy surrounds whether probiotics administered maternally or during infancy are healthful for allergic disease, and their use for autoimmunity is relatively unexplored. This review aims to discuss the use of maternal probiotics in health and autoimmune disease and to investigate their immunomodulatory properties. Preparation of this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number 1R01AR073240 and 1R15AR067418. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease with no known cure. Gut microbiota and the immune system have a close relationship that controls inflammation. Alterations in the gut microbiota, known as dysbiosis, can trigger SLE in both human and mouse. We previously published differences between the gut microbiota in lupus-prone MRL/Mp-Faslpr (lpr) mice compared to healthy controls, with lower Lactobacillaceae abundance in lpr mice. Restoring the gut microbiota with a mix of 5 different strains of this family significantly attenuated lupus-liked disease in lpr mice. In this study, we sought to understand the potential benefit of this mix in lpr mice with Cx3cr1 deficiency (KO). KO mice on C57BL/6 background display increased translocation of commensal bacteria into the mesenteric lymph node (MLN) and increased susceptibility to intestinal inflammation. However, whether CX3CR1+ macrophages play a role in controlling systemic inflammation was unclear. After backcrossing the KO allele to lpr mice, we found that KO lpr mice exhibited exacerbated splenomegaly and lymphadenopathy, as well as increased proteinuria. Interestingly, treating the KO lpr mice with the 5 Lactobacillus strains attenuated the enlargement of MLN and abrogated the increase of proteinuria. Furthermore, the effects of the probiotic bacteria were accompanied by downregulation of serum endotoxin, suggesting the reversal of a leaky gut that may explain their benefits on KO lpr mice. Together, our results show an important role of CX3CR1+ macrophages in controlling systemic inflammation in lupus, and that probiotic Lactobacillus spp. may be an effective treatment against SLE through preventing bacterial translocation.

Vitamin A deficiency (VAD) is observed in both human and mice with lupus nephritis (LN). However, whether VAD is a driving factor for LN progression is unclear. Here, we investigated the effect of VAD on the progression of LN in a lupus-prone mouse model, MRL/lpr. In these mice, while LN is not apparent before 8 weeks of age, the inflammation of lymphoid tissues suggesting initiation of lupus is evident at weaning. Thus, we initiated VAD either before or after weaning to reveal a potential time-dependent effect. At around 15 weeks of age with both types of VAD, we found exacerbated LN that was characterized by deteriorated kidney inflammation, impaired renal epithelial integrity, and dramatic squamous metaplasia of the renal pelvic urothelium. Both types of VAD provoked severe neutrophilic tubulointerstitial nephritis and accelerated renal failure. This was concomitant with significantly higher mortality in all VAD mice. Looking at an earlier time point of 7 weeks of age and before the onset of clinical LN, both types of VAD increased splenomegaly, lymphadenopathy, and circulating autoantibodies; three additional hallmarks of lupus, as well as renal infiltration of conventional and plasmacytoid dendritic cells. These results suggest VAD-driven deterioration of LN regardless of the time of VAD initiation. Our findings raise significant public health concerns that concurrent lupus and VAD may lead to more severe SLE. Future studies will elucidate the underlying mechanisms of how VAD modulates myeloid cells to accelerate the initiation of LN.

Hypersensitivity reactions and immune dysregulation have been reported with the use of quaternary ammonium compound disinfectants (QACs). We hypothesized that QAC exposure would exacerbate autoimmunity associated with systemic lupus erythematosus (lupus). Surprisingly, however, we found that compared to QAC-free mice, ambient exposure of lupus-prone mice to QACs led to smaller spleens with no change in circulating autoantibodies or the severity of glomerulonephritis. This suggests that QACs may have immunosuppressive effects on lupus. Using a microfluidic device, we showed that ambient exposure to QACs reduced directional migration of bone marrow-derived neutrophils toward an inflammatory chemoattractant ex vivo. Consistent with this, we found decreased infiltration of neutrophils into the spleen. While bone marrow-derived neutrophils appeared to exhibit a pro-inflammatory profile, upregulated expression of PD-L1 was observed on neutrophils that infiltrated the spleen, which in turn interacted with PD-1 on T cells and modulated their fate. Specifically, QAC exposure hindered activation of splenic T cells and increased apoptosis of effector T-cell populations. Collectively, these results suggest that ambient QAC exposure decreases lupus-associated splenomegaly likely through neutrophil-mediated toning of T-cell activation and/or apoptosis. However, our findings also indicate that even ambient exposure could alter immune cell phenotypes, functions, and their fate. Further investigations on how QACs affect immunity under steady-state conditions are warranted. National Institute of Arthritis and Musculoskeletal and Skin DiseasesUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Arthritis & Musculoskeletal & Skin Diseases (NIAMS); National Institute of Environmental Health SciencesUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Environmental Health Sciences (NIEHS) [T32-ES007032]; National Institute of General Medical Sciences of the National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of General Medical Sciences (NIGMS) [R35GM133610] Funding was received from National Institute of Arthritis and Musculoskeletal and Skin Diseases. We also would like to acknowledge the following funding sources. Environmental Pathology and Toxicology Training Grant funded by the National Institute of Environmental Health Sciences (T32-ES007032) for supporting RP. Seguin. The National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM133610 for supporting M Moarefian.

Systemic Lupus Erythematosus(SLE), a disease of various genetic and environmental origins, is often characterized by immune complexes causing inflammation and damage in various organ systems, such as the brain, kidneys, lung, joint, and skin. Current treatment for SLE include steroidal and nonsteroidal anti-inflammatory drugs, antimalarial agents, and immunosuppressive medications. These treatments primarily reduce symptoms, and patients may develop side effects with long-term care. The gut microbiome has an important role in various metabolic and immunological diseases. A previous study found that increased Lactobacillus colonization restored the mucosal barrier, and increased abundance in these bacteria attenuated SLE symptoms through reversing a ‘leaky gut’. This allows for potential therapies of SLE with probiotics. In preliminary trials, vancomycin treatment during active disease killed gram-positive bacteria, but spared Lactobacillus, and attenuated symptoms. Surprisingly, significantly decreased amounts of CD10+CD19+ B cells that produce IL-10 (B10) were found when vancomycin was given before the onset of lupus. Vancomycin treatment before disease onset significantly removed bacterial DNA from the gut and circulation, causing exacerbation of clinical symptoms. Oral gavage of activated B10 cells in vancomycin treated mice rescued the negative effects of vancomycin treated mice at the pre-disease stage. This work suggests that a form of oral tolerance induced by bacterial DNA-mediated expansion of B10 cells suppress disease onset in the autoimmune-prone MRL/lpr mouse model. Future studies are warranted to further define the mechanism behind bacterial DNA promoting B10 cells.

Maternal factors play an essential role in shaping the immune system of the newborn, yet it is unknown whether maternal factors could modulate the development of systemic lupus erythematosus (SLE) in the offspring. Activation-induced cytidine deaminase (AID) is an enzyme required for somatic hypermutation and class switch recombination. Given that IgG and IgA isotypes account for the vast majority of passive immunity in rodents, our previously established AID-deficient BXSB mice provide a model in which no IgG or IgA antibodies are transferred to the offspring. In this study, we compared genotypically identical mice born to either AID-sufficient dams or AID-deficient dams and evaluated the effects of maternal antibodies in disease progression. Offspring from knockout dams developed disease at a faster rate, as shown by more severe nephritis and elevated pathogenic autoantibodies compared to their counterparts born to wild type dams. In addition, the splenic structure was intensively disrupted with disorganized follicles and fewer discernible germinal centers. A more activated T cell phenotype was also characterized in maternal antibody-deficient mice. When immune competent pups were cross-fostered onto AID deficient dams, these mice exhibited more severe disease phenotypes, indicating the protective antibody effect contributes to the modulation of SLE progression in post-natal period. Overall, these findings highlight the importance of maternal antibodies in programming the immune system and altering the SLE development in the offspring.

Recently, we demonstrated that treatment with all- trans-retinoic acid (tRA) induced a paradoxical effect on immune activation during the development of autoimmune lupus. Here, we further describe its negative effects on mediating neuroinflammation and neurodegeneration. Female MRL/lpr mice were orally administered tRA or VARA (retinol mixed with 10% tRA) from 6 to 14 weeks of age. Both treatments had a significant effect on brain weight, which correlated with histopathological evidence of focal astrogliosis, meningitis, and ventriculitis. Infiltration of CD138- and Iba1-positve immune cells was observed in the third ventricle and meninges of treated mice that co-labeled with ICAM-1, indicating their inflammatory nature. Increased numbers of circulating plasma cells, autoantibodies, and total IgG were also apparent. IgG and C3 complement deposition in these brain regions were also prominent as was focal astrogliosis surrounding the ventricular lining and meninges. Using Fluoro-Jade staining, we further demonstrate that neuroinflammation was accompanied by neurodegeneration in the cortex of treated mice compared with vehicle controls. These findings indicate that vitamin A exposure exacerbates the immunogenic environment of the brain during the onset of systemic autoimmune disease. Vitamin A may therefore compromise the immuno-privileged nature of the central nervous system under a predisposed immunogenic environment.

We sought to determine if a selective HDAC6 inhibitor (ACY-738) decreases disease in NZB/W mice. From 22 to 38weeks-of-age, mice were injected intraperitoneally with 5 or 20mg/kg of ACY-738, or vehicle control. Body weight and proteinuria were measured every 2weeks, while sera anti-dsDNA, Ig isotypes, and cytokine levels were measured every 4weeks. Kidney disease was determined by evaluation of sera, urine, immune complex deposition, and renal pathology. Flow cytometric analysis assessed thymic, splenic, bone marrow, and peripheral lymphocyte differentiation patterns. Our results showed HDAC6 inhibition decreased SLE disease by inhibiting immune complex-mediated glomerulonephritis, sera anti-dsDNA levels, and inflammatory cytokine production and increasing splenic Treg cells. Inhibition of HDAC6 increased the percentage of cells in the early-stage developmental fractions of both pro- and pre-B cells. These results suggest that specific HDAC6 inhibition may be able to decrease SLE disease by altering aberrant T and B cell differentiation.

We previously showed that all-trans-retinoic acid (tRA) exacerbated pre-existing autoimmunity in lupus; however, its effects before disease onset are unknown. Here, using a pristane-induced model, we show that tRA exerts tissue-specific effects when given at the initiation vs. continuation phase of lupus. Pre-pristane treatment with tRA aggravated glomerulonephritis through increasing renal expression of pro-fibrotic protein laminin β1, activating bone marrow CD11b- conventional dendritic cells, increasing the splenic CD4:CD8 ratio, and upregulating the interaction of ICAM-1 and LFA-1 that led to the infiltration of inflammatory cells to the spleen. Transcriptomic analysis revealed that prior to lupus induction, tRA significantly upregulated genes associated with cell differentiation, activation, and migration. Moreover, compared to pristane alone, tRA pre-treatment potentiated, whereas tRA post-treatment significantly suppressed, the renal expression of proinflammatory TNF-α, IL-1β, CCL2, and CCL3. While it may benefit the kidney, post-pristane treatment with tRA worsened the onset and severity of pristane-induced arthritis through promoting neutrophil and mononuclear cell infiltration into the joints. Together, these findings suggest that tRA supplementation regardless of the time of administration promotes chemokine-driven migration and tissue-specific infiltration of inflammatory cells, thereby exacerbating lupus-associated kidney or joint inflammation. Interestingly, both pre- and post-treatments with tRA modulated the gut microbiota in a similar fashion, suggesting a gut microbiota-independent mechanism by which tRA affects the initiation vs. continuation phase of lupus.

Dysbiosis in the gut microbiota has been observed in a various autoimmune disease, including SLE, which could cause a leaky gut, triggering an immune response, and thus worsening autoimmune disease expression. In our current studies, we hypothesized that increasing dietary fiber would create a healthy microbiota environment in the gut, leading to decreased leakiness of the gut and to decreased disease expression in and NZB/NZW female mice. NZB/NZW mice were placed on standardized high fiber (HF 30%) or low fiber (LF 0.4%) for 12 weeks beginning at 10 weeks of age. Mice were assessed as they aged for various parameters of disease including proteinuria and anti-dsDNA antibody production. Alteration of the microbiota and short chain fatty acid levels were also assessed. At 36 weeks-of-age, the mice were euthanized and we assessed occlusion protein expression, splenocyte profiles, and kidney tissue. We found as the mice aged, their body weights, anti-dsDNA antibody levels, and proteinuria were not significantly different between the groups. Similarly, there were no differences in SCFA levels. In regard to the microbiota, Chlostridiales bacteria were consistently increased in the HF treated mice compared to the LF treated mice. In regard to disease progression, spleen weight, immune cell profiles, proteinuria, dsDNA levels, and kidney pathology, there was no difference between the HF and LF treated groups. Taken together, these results indicate that in the NZB/W female mouse, a HF diet may alter the microbiota but does not influence disease progression.

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease with no known cure. The role of gut microbiota and its interaction with the immune system can direct tolerance induction to self-antigens not only in the intestinal mucosa but also at the systemic level. Our lab previously showed differences between the gut microbiota in lupus-prone MRL/Mp-Faslpr (lpr) mice compared to healthy controls, with lower Lactobacillaceae levels in lpr mice. Restoring the microbiota with a mix of 5 different Lactobacillus strains, L. reuteri, L. oris, L. johnsonii, L. gasseri, and L. rhamnosus significantly attenuated lupus-liked disease in lpr mice, by increasing regulatory T cells and balancing T helper-17 cells. Interestingly, we found L. reuteri and an uncultured Lactobacillus spp. were present based on 16S rRNA sequencing. To evaluate the effect of the main candidate L. reuteri in attenuating lupus, we treated lpr mice with the single bacterial strain. Surprisingly, L. reuteri did not reproduce the same results as the Lactobacillus mixture. Thus, we tested the remaining strains individually but none was able to recapitulate the effect of the 5 strains. Additionally, we treated lpr mice with the supernatant of the 5 strains containing secreted metabolites, which did not significantly attenuate lupus. These results suggest that communications among different strains of lactobacilli may be required in ameliorating the disease. Probiotic Lactobacillus spp. may be an alternative treatment against SLE, but further investigations are warranted to delineate the mechanisms of how multiple strains of lactobacilli can exert a beneficial effect as a community.

A leaky gut has been observed in a number of autoimmune diseases including type 1 diabetes, multiple sclerosis, inflammatory bowel disease, and systemic lupus erythematosus. Previous studies from our laboratory have shown that lupus mice also bear a leaky gut and that the intestinal barrier function can be enhanced by gut colonization of probiotics such as Lactobacillus spp. Retinoic acid (RA) can increase the relative abundance of Lactobacillus spp. in the gut. Interestingly, RA has also been shown to strengthen the barrier function of epithelial cells in vitro and in the absence of probiotic bacteria. These reports bring up an interesting question of whether RA exerts protective effects on the intestinal barrier directly or through regulating the microbiota colonization. In this review, we will discuss the roles of RA in immunomodulation, recent literature on the involvement of a leaky gut in different autoimmune diseases, and how RA shapes the outcomes of these diseases. Published version

A leaky gut has been observed in a number of autoimmune diseases including type 1 diabetes, multiple sclerosis, inflammatory bowel disease, and systemic lupus erythematosus. Previous studies from our laboratory have shown that lupus mice also bear a leaky gut and that the intestinal barrier function can be enhanced by gut colonization of probiotics such as Lactobacillus spp. Retinoic acid (RA) can increase the relative abundance of Lactobacillus spp. in the gut. Interestingly, RA has also been shown to strengthen the barrier function of epithelial cells in vitro and in the absence of probiotic bacteria. These reports bring up an interesting question of whether RA exerts protective effects on the intestinal barrier directly or through regulating the microbiota colonization. In this review, we will discuss the roles of RA in immunomodulation, recent literature on the involvement of a leaky gut in different autoimmune diseases, and how RA shapes the outcomes of these diseases.