Gut Microorganisms and Autism: the Latest Research

LPS and Autism

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Treatments for LPS

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LPS and the Brain LPS and Thyroid LPS and GI System LPS and Immunity Other Effects of LPS
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How LPS affects the IMMUNE System

http://www.thoughtfulhouse.org/0405-conf-pashwood.htm

Paul Ashwood, a professor at the M.I.N.D Institute, lectured on the immune systems of ASD children at Thoughtful House. He noted many differences between the immune systems of typical children and ASD children. Exposure to Lipopolysaccharides (LPS) explains most of these differences. Research articles on this web page indicate that some features of the autistic immune system reported by the M.I.N.D. Institute are due to LPS.

These features include the following:

There were increases in NK cells and monocytes
Increases in lymphocytes
Altered or inappropriate cytokine production
Increase in TNF alpha.
(These differences were also mentioned in Paul Ashwood's lecture.)

Paul Ashwood concluded his presentation by saying that there is a possibility that microorganisms might be involved in the dysregulation of the immune system.
"Various stimulators may be involved, such as pathogenic agents and molecules that are not usually present under physiological conditions (e.g, viruses, bacteria, neuroactive molecules from the diet)."


The following research articles demonstrate the influence of LPS on the immune system.

Altered or inappropriate cytokine production upon stimulation

View this article in PubMed

1: Immunobiology. 1993 Apr;187(3-5):403-16.

Response of man to endotoxin.

Martich GD, Boujoukos AJ, Suffredini AF.

Critical Care Medicine Department, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD.

Endotoxin, a cell wall component of Gram-negative bacteria, plays a central role in the pathogenesis of septic shock. By administering small doses of intravenous endotoxin to humans, a variety of acute inflammatory responses are induced which are qualitatively similar to those that occur during the early stages of septic shock. Within hours of the administration of intravenous endotoxin to human volunteers, changes occur in systemic hemodynamics, ventricular function, pulmonary gas exchange and permeability. In conjunction with these changes in organ function, a wide variety of inflammatory mediators are released which appear to contribute to these responses. These include the release of proinflammatory cytokines (e.g. tumor necrosis factor-alpha, IL-1 beta, IL-6, IL-8), activation of the fibrinolytic system, kallikrein-kinin generation and phospholipase A2 release. Phagocytic leukocytes are primed for enhanced inflammatory responses following endotoxin administration. Counter-regulatory responses are initiated in parallel and may serve to limit some of the end-organ responses by the inflammatory mediators. This human model provides a unique opportunity to extend previous concepts of acute inflammation and to evaluate the earliest responses activated after exposure to an important bacterial component. Defining the pathways and responses initiated during acute human endotoxemia may allow a better understanding of host responses that are critical to the development of organ dysfunction and shock due to severe infections.

PMID: 8330905 [PubMed - indexed for MEDLINE]


Increases in NK (Natural Killer ) cells.

View this article in PubMed
J Immunol. 1996 Apr 1;156(7):2436-42.

LPS induces NK1.1+ alpha beta T cells with potent cytotoxicity in the liver of mice via production of IL-12 from Kupffer cells.

Takahashi M, Ogasawara K, Takeda K, Hashimoto W, Sakihara H, Kumagai K, Anzai R, Satoh M, Seki S.

Department of Oral Surgery, Tohku University School of Dentistry, Sendai, Japan.

We recently reported that systemic administration of IL-12 into mice activates NK1.1+ alpha beta T cells with intermediate TCR (NK1+TCRint) and induces strong MHC-unrestricted cytotoxicity in C57BL/6 mice. In the present report, we examined the effect of LPS on Kupffer cells and NK1+TCRint, cells in C57BL/6 mice. Administration of LPS, as well as synthetic lipid A analogue (ONO-4007), but not detoxified LPS, induces the increase of NK1 expression of NK1+TCRint cells (NKlhighTCRint) and the acquisition of strong MHC-unrestricted cytotoxicity of these cells against NK-sensitive and NK-resistant targets as does IL-12 administration. LPS as well as ONO-4007 induced IL-12 mRNA in hepatic mononuclear cells, mainly in plastic-adherent Kupffer cells. LPS-induced cytotoxicity of hepatic mononuclear cells was greatly reduced by in vivo injections of anti-IL-12 Ab, to a lesser extent by anti-IFN-gamma Ab, but not by anti-IL-1 nor anti-TNF-alpha Ab. Pretreatment of mice with LPS induced inhibition of hepatic metastases of i.v. injected EL4 cells in C57BL/6 euthymic and athymic mice and this antimetastasis was inhibited by injection of anti-IL-12 Ab. This antimetastatic effect of LPS in the liver was also observed in different strains of mice and tumors, In contrast to IL-12, however, LPS was not so effective when administered after tumor inoculation. These results revealed that LPS (lipid A) stimulates NK1+TCRint cells through IL-12 production from Kupffer cells and suggest that bacterial components, probably including those from intestine, are activators of Kupffer cells and NK1+TCRint, cells in the liver. It is also suggested that the host condition as well as LPS-induced cytokines other than IL-12 may affect antitumor effect induced by LPS in the liver.

PMID: 8786302 [PubMed - indexed for MEDLINE]


LPs produces an increase in lymphocytes

View this article in PubMed
1: Eur Respir J. 1992 Sep;5(8):992-6.

Lipopolysaccharide (LPS) inhalation in healthy subjects increases neutrophils, lymphocytes and fibronectin levels in bronchoalveolar lavage fluid.

Sandstrom T, Bjermer L, Rylander R.

Dept of Lung Medicine, University Hospital of Umea, Sweden.

Bacterial endotoxin has been suggested as responsible for the development of subjective symptoms and transient or chronic lung function impairment seen after exposure to organic dusts in cotton mills, poultry houses, swine confinement buildings and saw mills. Animal experiments have demonstrated bronchoalveolar neutrophilia being the most prominent cell response in animals following bacterial lipopolysaccharide (LPS) inhalation. The present study was conducted to obtain information on some aspects of the early inflammatory response to inhaled LPS in man. Eight healthy nonsmoking subjects, 23-27 yrs old, underwent bronchoalveolar lavage (BAL), 3 h after a provocation test with 100 micrograms LPS from E. coli dissolved in 2 ml isotonic NaCl. The solution was aerosolized with a jet nebulizer and inhaled. The calculated dose delivered to the lung was approximately 25 micrograms, which equals exposure in some occupational settings. The BAL data for each individual subject were compared with data from a control BAL performed at least 6 weeks prior to the LPS challenge. The major cellular response to LPS, reflected in BAL fluid, was an approximately hundredfold increase in neutrophils. The total number of lymphocytes was on average tripled. The alveolar macrophage phagocytosis of opsonized yeast particles in vitro was significantly reduced. A further indicator of an ongoing inflammation was an increase in fibronectin. No changes were seen in the levels of BAL albumin, indicating that the elevated level of fibronectin could not be explained by an increased permeability, but rather by a local production. The results correspond with data from animal studies and further supports the hypothesis that bacterial LPS is important in the pulmonary reaction induced by organic dusts.

PMID: 1426208 [PubMed - indexed for MEDLINE]

Stimulation of Human T Lymphocytes by LPS
http://www.jimmunol.org/cgi/content/abstract/160/7/3412
The Journal of Immunology, 1998, 160: 3412-3418.
Copyright 1998 by The American Association of Immunologists

Stimulation of Human T Lymphocytes by LPS Is MHC Unrestricted, But Strongly Dependent on B7 Interactions1

Taila Mattern*, Hans-Dieter Flad*, Lore Brade, Ernst T. Rietschel and Artur J. Ulmer2,*

* Departments of Immunology and Cell Biology and Immunochemistry and Biochemical Microbiology, Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany

Recently, we have shown that LPS is a potent inducer of human T cell proliferation and lymphokine production. However, the activation of T cells by LPS has been demonstrated to be monocyte dependent and to require direct cell-to-cell contact. Here, we investigated the role of monocytes as accessory cells and the requirement for costimulatory signals in more detail. We found that the accessory cell activity of monocytes during LPS-induced T cell proliferation is characterized by the following features: LPS-primed monocytes are competent stimulators of T cell proliferation; interaction of LPS with monocytes during the priming step is dependent on CD14 and is sensitive to ammonia; monocyte/T cell interactions are not MHC restricted but are strongly dependent on interactions of CD28 and/or CTLA-4 on T cells and their ligands CD80 and/or CD86 on monocytes. CD80 seems to be crucial for the activation of T cells by monocytes, since monocytes expressing CD86 but not CD80 after LPS stimulation were unable to stimulate T cells; IL-12, at least as a costimulatory factor, but not IL-15, is important in LPS-induced T cell proliferation. Taken together, our results indicate that LPS acts neither as a mitogen, nor as a superantigen, nor as an Ag. The activation of human T cells by LPS requires the help of accessory functions by primed monocytes and is MHC unrestricted but needs costimulatory signals via CD28 and/or CTLA-4.


Increase in TNF alpha.

The two articles below show that LPS brings an increase in TNF alpha for both mice and children with autism.

View this article in PubMed
1: J Neuroimmunol. 2001 Nov 1;120(1-2):170-9.

Proinflammatory and regulatory cytokine production associated with innate and adaptive immune responses in children with autism spectrum disorders and developmental regression.

Jyonouchi H, Sun S, Le H.

Department of Pediatrics, University of Minnesota, MMC 610 FUMC, 420 Delaware Street SE, Minneapolis, MN 55455, USA. jyono001@tc.umn.edu

We determined innate and adaptive immune responses in children with developmental regression and autism spectrum disorders (ASD, N=71), developmentally normal siblings (N=23), and controls (N=17). With lipopolysaccharide (LPS), a stimulant for innate immunity, peripheral blood mononuclear cells (PBMCs) from 59/71 (83.1%) ASD patients produced >2 SD above the control mean (CM) values of TNF-alpha, IL-1beta, and/or IL-6 produced by control PBMCs. ASD PBMCs produced higher levels of proinflammatory/counter-regulatory cytokines without stimuli than controls. With stimulants of phytohemagglutinin (PHA), tetanus, IL-12p70, and IL-18, PBMCs from 47.9% to 60% of ASD patients produced >2 SD above the CM values of TNF-alpha depending on stimulants. Our results indicate excessive innate immune responses in a number of ASD children that may be most evident in TNF-alpha production.

PMID: 11694332 [PubMed - indexed for MEDLINE]

View this article in PubMed
Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration.

* Qin L, * Wu X, * Block ML, * Liu Y, * Breese GR, * Hong JS, * Knapp DJ, * Crews FT.

Bowles Center for Alcohol Studies, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Inflammation is implicated in the progressive nature of neurodegenerative diseases, such as Parkinson's disease, but the mechanisms are poorly understood. A single systemic lipopolysaccharide (LPS, 5 mg/kg, i.p.) or tumor necrosis factor alpha (TNFalpha, 0.25 mg/kg, i.p.) injection was administered in adult wild-type mice and in mice lacking TNFalpha receptors (TNF R1/R2(-/-)) to discern the mechanisms of inflammation transfer from the periphery to the brain and the neurodegenerative consequences. Systemic LPS administration resulted in rapid brain TNFalpha increase that remained elevated for 10 months, while peripheral TNFalpha (serum and liver) had subsided by 9 h (serum) and 1 week (liver). Systemic TNFalpha and LPS administration activated microglia and increased expression of brain pro-inflammatory factors (i.e., TNFalpha, MCP-1, IL-1beta, and NF-kappaB p65) in wild-type mice, but not in TNF R1/R2(-/-) mice. Further, LPS reduced the number of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra (SN) by 23% at 7-months post-treatment, which progressed to 47% at 10 months. Together, these data demonstrate that through TNFalpha, peripheral inflammation in adult animals can: (1) activate brain microglia to produce chronically elevated pro-inflammatory factors; (2) induce delayed and progressive loss of DA neurons in the SN. These findings provide valuable insight into the potential pathogenesis and self-propelling nature of Parkinson's disease. (c) 2007 Wiley-Liss, Inc.

PMID: 17203472 [PubMed - indexed for MEDLINE]


Increase in monocytes

View this article in PubMed
PMID: 17291629 [PubMed - in process]
1: Mol Pharmacol. 2007 Mar 14; [Epub ahead of print]
Inhibition of TNF-{alpha} through Selective Blockade of Pre-mRNA Splicing by Shikonin.

* Chiu SC, * Yang NS.

Graduate Institute of Life Sciences, National Defense Medical Center.

We previously developed a gene-gun-based in vivo screening system and identified shikonin as a potent suppressor of tumor necrosis factor-alpha (TNF-alpha) gene expression. Here, we show that shikonin selectively inhibits the expression of TNF-alpha at the RNA splicing level. Treatment of lipopolysaccharide- stimulated human primary monocytes and THP-1 cells with shikonin resulted in normal transcriptional induction of TNF-alpha, but unspliced pre-mRNA accumulated at the expense of functional mRNA. This effect occurred with noncytotoxic doses of shikonin and was highly specific, because mRNA production of neither a housekeeping gene nor another inflammatory cytokine gene, interleukine-8 (IL-8), was affected. Moreover, co-treatment with LPS and shikonin increased the end-point protein production of IL-8, accompanied by suppressed activation of the dsRNA-activated protein kinase (PKR) pathway. Because PKR inactivation has been shown to downregulate the splicing process of TNF-alpha RNA and interfere with translation, our findings suggest that shikonin may achieve differential modulation of cytokine protein expression through inactivation of the PKR pathway, and reveal that regulation of TNF-alpha pre-mRNA splicing may constitute a promising target for future anti- inflammatory application.

PMID: 17360831 [PubMed - as supplied by publisher]


View this article in PubMed
1: J Biomed Mater Res B Appl Biomater. 2007 Feb 6; [Epub ahead of print] Cytokine secretion from monocytes persists differentially after activator removal-One mechanism of long-term biological response to implants.

* Messer RL, * Lewis JB, * Wataha JC, * Adams Y, * Tseng WY.

Department of Oral Biology and Maxillofacial Pathology, Medical College of Georgia, Augusta, Georgia.

Biomedical implants significantly improve the quality of life in an ever-increasing number of patients. However, inflammation of tissues around implants remains a long-term, post-placement sequelae that may contribute to implant failure. Infection-mediated failure is partly a consequence of inappropriate host response and chronic inflammation, and is mediated primarily by the secretory products of monocytes and macrophages. Although the secretion of inflammatory mediators from activated monocytes is well characterized, the resolution of mediator levels post-activation is relatively unstudied. The current study defines the time course of cytokine secretion by activated human monocytes after the activator has been removed. THP1 human monocytes were activated by LPS, and cytokine secretion was monitored over time after LPS removal using enzyme-linked immunosorbent assays (TNFalpha or IL8) or a cytokine array. The release of cytokines was compared with conditions without LPS removal. As expected, secretion of nearly all cytokines was reduced when LPS was removed, but the amount of the reduction was highly cytokine-dependent. Furthermore, levels of cytokines were stable in medium alone but not in cell-culture, suggesting an active process to either degrade or internalize secreted cytokines. Our results are consistent with clinical experience that inflammation resolves rapidly after treatment to remove bacteria or inflamed tissue. However, the differential cytokine regulation indicates a sophisticated coordination of cytokine levels probably associated with management of the wound healing response after removal of the bacterial insult. This wound healing response is one critical component of the long-term success of biomedical implants. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2007.

PMID: 17285613 [PubMed - as supplied by publisher]


View this article in PubMed
1: Neuropsychobiology. 2002;45(1):1-6.

Activation of the inflammatory response system in autism.

Croonenberghs J, Bosmans E, Deboutte D, Kenis G, Maes M.

University Center of Child and Adolescent Psychiatry, Antwerp, Belgium.

BACKGROUND/AIM: There is now some evidence that autism may be accompanied by abnormalities in the inflammatory response system (IRS). Products of the IRS, such as proinflammatory cytokines, may induce some of the behavioral symptoms of autism, such as social withdrawal, resistance to novelty and sleep disturbances. The main aim of the present study was to examine whether autism is accompanied by an activation of the IRS. METHODS: We measured the production of interleukin (IL)-6, IL-10, the IL-1 receptor antagonist (IL-1RA), interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha by whole blood and the serum concentrations of IL-6, the IL-2 receptor (IL-2R) and IL-1RA. RESULTS: This study showed a significantly increased production of IFN-gamma and IL-1RA and a trend toward a significantly increased production of IL-6 and TNF-alpha by whole blood of autistic children. There were no significant differences in the serum concentrations of IL-6, IL-2R and IL-1RA between autistic and normal children. CONCLUSIONS: These results suggest that autism may be accompanied by an activation of the monocytic (increased IL-1RA) and Th-1-like (increased IFN-gamma) arm of the IRS. It is hypothesized that increased production of proinflammatory cytokines could play a role in the pathophysiology of autism. Copyright 2002 S. Karger AG, Basel

PMID: 11803234 [PubMed - indexed for MEDLINE]