Associations of Mucosal Nerve Fiber Innervation Density with Hirschsprung-Associated Enterocolitis: A Retrospective Three-Center Cohort Study.

OBJECTIVE: Hirschsprung's disease (HSCR) is a congenital intestinal neurodevelopmental disorder characterized by the absence of enteric ganglion cells in the distal colon. Although Hirschsprung-associated enterocolitis (HAEC) is the most frequent life-threatening complication in HSCR, to date reliable biomarkers predicting the likelihood of HAEC are yet to be established. We established a three-center retrospective study including 104 HSCR patients surgically treated between 1998 and 2019. MATERIALS AND METHODS: Patient-derived cryopreserved or paraffin-preserved colonic tissue at surgery was analyzed via ßIII-tubulin immunohistochemistry. We subsequently determined extrinsic mucosal nerve fiber density in resected rectosigmoid specimens and classified HSCR patients accordingly into nerve fiber-high or fiber-low groups. We compared the distribution of clinical parameters obtained from medical records between the fiber-high (n = 36) and fiber-low (n = 68) patient groups. We assessed the association between fiber phenotype and enterocolitis using univariate and multivariate logistic regression adjusted for age at operation. RESULTS: Enterocolitis was more prevalent in patients with sparse mucosal nerve fiber innervation (fiber-low phenotype, 87%) compared with the fiber-high phenotype (13%; p = 0.002). In addition, patients developing enterocolitis had a younger age at surgery (3 vs. 7 months; p = 0.016). In the univariate analysis, the odds for enterocolitis development in the fiber-low phenotype was 5.26 (95% confidence interval [CI], 1.67-16.59; p = 0.005) and 4.01 (95% CI, 1.22-13.17; p = 0.022) when adjusted for age. CONCLUSION: Here, we showed that HSCR patients with a low mucosal nerve fiber innervation grade in the distal aganglionic colon have a higher risk of developing HAEC. Consequently, histopathologic analysis of the nerve fiber innervation grade could serve as a novel sensitive prognostic marker associated with the development of enterocolitis in HSCR patients.

Lack of Mucosal Cholinergic Innervation Is Associated With Increased Risk of Enterocolitis in Hirschsprung's Disease.

BACKGROUND & AIMS: Hirschsprung's disease (HSCR) is a congenital intestinal motility disorder defined by the absence of enteric neuronal cells (ganglia) in the distal gut. The development of HSCR-associated enterocolitis remains a life-threatening complication. Absence of enteric ganglia implicates innervation of acetylcholine-secreting (cholinergic) nerve fibers. Cholinergic signals have been reported to control excessive inflammation, but the impact on HSCR-associated enterocolitis is unknown. METHODS: We enrolled 44 HSCR patients in a prospective multicenter study and grouped them according to their degree of colonic mucosal acetylcholinesterase-positive innervation into low-fiber and high-fiber patient groups. The fiber phenotype was correlated with the tissue cytokine profile as well as immune cell frequencies using Luminex analysis and fluorescence-activated cell sorting analysis of colonic tissue and immune cells. Using confocal immunofluorescence microscopy, macrophages were identified in close proximity to nerve fibers and characterized by RNA-seq analysis. Microbial dysbiosis was analyzed in colonic tissue using 16S-rDNA gene sequencing. Finally, the fiber phenotype was correlated with postoperative enterocolitis manifestation. RESULTS: The presence of mucosal nerve fiber innervation correlated with reduced T-helper 17 cytokines and cell frequencies. In high-fiber tissue, macrophages co-localized with nerve fibers and expressed significantly less interleukin 23 than macrophages from low-fiber tissue. HSCR patients lacking mucosal nerve fibers showed microbial dysbiosis and had a higher incidence of postoperative enterocolitis. CONCLUSIONS: The mucosal fiber phenotype might serve as a prognostic marker for enterocolitis development in HSCR patients and may offer an approach to personalized patient care and new therapeutic options.

Cholinergic Signaling Attenuates Pro-Inflammatory Interleukin-8 Response in Colonic Epithelial Cells.

Infants affected by Hirschsprung disease (HSCR), a neurodevelopmental congenital disorder, lack ganglia of the intrinsic enteric nervous system (aganglionosis) in a variable length of the colon, and are prone to developing severe Hirschsprung-associated enterocolitis (HAEC). HSCR patients typically show abnormal dense innervation of extrinsic cholinergic nerve fibers throughout the aganglionic rectosigmoid. Cholinergic signaling has been reported to reduce inflammatory response. Consequently, a sparse extrinsic cholinergic innervation in the mucosa of the rectosigmoid correlates with increased inflammatory immune cell frequencies and higher incidence of HAEC in HSCR patients. However, whether cholinergic signals influence the pro-inflammatory immune response of intestinal epithelial cells (IEC) is unknown. Here, we analyzed colonic IEC isolated from 43 HSCR patients with either a low or high mucosal cholinergic innervation density (fiber-low versus fiber-high) as well as from control tissue. Compared to fiber-high samples, IEC purified from fiber-low rectosigmoid expressed significantly higher levels of IL-8 but not TNF-α, IL-10, TGF-ß1, Muc-2 or tight junction proteins. IEC from fiber-low rectosigmoid showed higher IL-8 protein concentrations in cell lysates as well as prominent IL-8 immunoreactivity compared to IEC from fiber-high tissue. Using the human colonic IEC cell line SW480 we demonstrated that cholinergic signals suppress lipopolysaccharide-induced IL-8 secretion via the alpha 7 nicotinic acetylcholine receptor (a7nAChR). In conclusion, we showed for the first time that the presence of a dense mucosal cholinergic innervation is associated with decreased secretion of IEC-derived pro-inflammatory IL-8 in the rectosigmoid of HSCR patients likely dependent on a7nAChR activation. Owing to the association between IL-8 and enterocolitis-prone, fiber-low HSCR patients, targeted therapies against IL-8 might be a promising immunotherapy candidate for HAEC treatment.

Gut microbiota modulate T cell trafficking into human colorectal cancer.

OBJECTIVE: Tumour-infiltrating lymphocytes (TILs) favour survival in human colorectal cancer (CRC). Chemotactic factors underlying their recruitment remain undefined. We investigated chemokines attracting T cells into human CRCs, their cellular sources and microenvironmental triggers. DESIGN: Expression of genes encoding immune cell markers, chemokines and bacterial 16S ribosomal RNA (16SrRNA) was assessed by quantitative reverse transcription-PCR in fresh CRC samples and corresponding tumour-free tissues. Chemokine receptor expression on TILs was evaluated by flow cytometry on cell suspensions from digested tissues. Chemokine production by CRC cells was evaluated in vitro and in vivo, on generation of intraperitoneal or intracecal tumour xenografts in immune-deficient mice. T cell trafficking was assessed on adoptive transfer of human TILs into tumour-bearing mice. Gut flora composition was analysed by 16SrRNA sequencing. RESULTS: CRC infiltration by distinct T cell subsets was associated with defined chemokine gene signatures, including CCL5, CXCL9 and CXCL10 for cytotoxic T lymphocytes and T-helper (Th)1 cells; CCL17, CCL22 and CXCL12 for Th1 and regulatory T cells; CXCL13 for follicular Th cells; and CCL20 and CCL17 for interleukin (IL)-17-producing Th cells. These chemokines were expressed by tumour cells on exposure to gut bacteria in vitro and in vivo. Their expression was significantly higher in intracecal than in intraperitoneal xenografts and was dramatically reduced by antibiotic treatment of tumour-bearing mice. In clinical samples, abundance of defined bacteria correlated with high chemokine expression, enhanced T cell infiltration and improved survival. CONCLUSIONS: Gut microbiota stimulate chemokine production by CRC cells, thus favouring recruitment of beneficial T cells into tumour tissues.

Water permeability is a measure of severity in acute appendicitis.

Acute appendicitis is the most common indication for pediatric abdominal emergency surgery. Determination of the severity of appendicitis on clinical grounds is challenging. Complicated appendicitis presenting with perforation, abscess or diffuse peritonitis is not uncommon. The question remains why and when acute appendicitis progresses to perforation. The aim of this study was to assess the impact of water permeability on the severity of appendicitis. We show that AQP1 expression and water permeability in appendicitis correlate with the stage of inflammation and systemic infection parameters, leading eventually to perforation of the appendix. AQP1 is also expressed within the ganglia of the enteric nervous system and ganglia count increases with inflammation. Severity of appendicitis can be correlated with water permeability measured by AQP1 protein expression and increase of ganglia count in a progressive manner. This introduces the question if regulation of water permeability can present novel curative or ameliorating therapeutic options.

A minimum number of autoimmune T cells to induce autoimmunity?

While autoimmune T cells are present in most individuals, only a minority of the population suffers from an autoimmune disease. To better appreciate the limits of T cell tolerance, we carried out experiments to determine how many autoimmune T cells are required to initiate an experimental autoimmune disease. Variable numbers of autoimmune OT-I T cells were transferred into RIP-OVA mice, which were injected with antigen-loaded DCs in a single footpad; this restricted T cell priming to a few OT-I T cells that are present in the draining popliteal lymph node. Using selective plane illumination microscopy (SPIM) we counted the number of OT-I T cells present in the popliteal lymph node at the time of priming. Analysis of our data suggests that a single autoimmune T cell cannot induce an experimental autoimmune disease, but a "quorum" of 2-5 autoimmune T cells clearly has this capacity.

Antigen affinity and antigen dose exert distinct influences on CD4 T-cell differentiation.

Cumulative T-cell receptor signal strength and ensuing T-cell responses are affected by both antigen affinity and antigen dose. Here we examined the distinct contributions of these parameters to CD4 T-cell differentiation during infection. We found that high antigen affinity positively correlates with T helper (Th)1 differentiation at both high and low doses of antigen. In contrast, follicular helper T cell (TFH) effectors are generated after priming with high, intermediate, and low affinity ligand. Unexpectedly, memory T cells generated after priming with very low affinity antigen remain impaired in their ability to generate secondary Th1 effectors, despite being recalled with high affinity antigen. These data challenge the view that only strongly stimulated CD4 T cells are capable of differentiating into the TFH and memory T-cell compartments and reveal that differential strength of stimulation during primary T-cell activation imprints unique and long lasting T-cell differentiation programs.

Gain of function in the immune system caused by a ryanodine receptor 1 mutation.

Mutations in RYR1, the gene encoding ryanodine receptor 1, are linked to a variety of neuromuscular disorders including malignant hyperthermia (MH), a pharmacogenetic hypermetabolic disease caused by dysregulation of Ca(2+) in skeletal muscle. RYR1 encodes a Ca(2+) channel that is predominantly expressed in skeletal muscle sarcoplasmic reticulum, where it is involved in releasing the Ca(2+) necessary for muscle contraction. Other tissues, however, including cells of the immune system, have been shown to express ryanodine receptor 1; in dendritic cells its activation leads to increased surface expression of major histocompatibility complex II molecules and provides synergistic signals leading to cell maturation. In the present study, we investigated the impact of an MH mutation on the immune system by studying the RYR1Y522S knock-in mouse. Our results show that there are subtle but significant differences both in resting 'non-challenged' mice as well as in mice treated with antigenic stimuli, in particular the knock-in mice: (i) have dendritic cells that are more efficient at stimulating T cell proliferation, (ii) have higher levels of natural IgG1 and IgE antibodies, and (iii) are faster and more efficient at mounting a specific immune response in the early phases of immunization. We suggest that some gain-of-function MH-linked RYR1 mutations might offer selective immune advantages to their carriers. Furthermore, our results raise the intriguing possibility that pharmacological activation of RyR1 might be exploited for the development of new classes of vaccines and adjuvants.

Absence of TRIF signaling in lipopolysaccharide-stimulated murine mast cells.

In macrophages, two signaling pathways, dependent on MyD88 or TIR domain-containing adaptor-inducing IFN-ß (TRIF) signaling, emanate from the LPS receptor TLR4/MD-2. In this study, we show that in murine bone marrow-derived mast cells (BMMCs), only the MyD88-dependent pathway is activated by LPS. The TRIF signaling branch leading both to NF-κB activation and enhanced proinflammatory cytokine production, as well as to IRF3 activation and subsequent IFN-ß production, is absent in LPS-stimulated BMMCs. IRF3 activation is also absent in peritoneal mast cells from LPS-injected mice. We observed strongly diminished TRAM expression in BMMCs, but overexpression of TRAM only moderately enhanced IL-6 and did not boost IFN-ß responses to LPS in these cells. A combination of very low levels of TRAM and TLR4/MD-2 with the known absence of membrane-bound CD14 are expected to contribute to the defective TRIF signaling in mast cells. We also show that, unlike in macrophages, in BMMCs the TRIF-dependent and -independent IFN-αß responses to other recognized IFN inducers (dsRNA, adenovirus, and B-DNA) are absent. These results show how the response to the same microbial ligand using the same receptor can be regulated in different cell types of the innate immune system.

Crucial role for human Toll-like receptor 4 in the development of contact allergy to nickel.

Allergies to nickel (Ni(2+)) are the most frequent cause of contact hypersensitivity (CHS) in industrialized countries. The efficient development of CHS requires both a T lymphocyte-specific signal and a proinflammatory signal. Here we show that Ni(2+) triggered an inflammatory response by directly activating human Toll-like receptor 4 (TLR4). Ni(2+)-induced TLR4 activation was species-specific, as mouse TLR4 could not generate this response. Studies with mutant TLR4 proteins revealed that the non-conserved histidines 456 and 458 of human TLR4 are required for activation by Ni(2+) but not by the natural ligand lipopolysaccharide. Accordingly, transgenic expression of human TLR4 in TLR4-deficient mice allowed efficient sensitization to Ni(2+) and elicitation of CHS. Our data implicate site-specific human TLR4 inhibition as a potential strategy for therapeutic intervention in CHS that would not affect vital immune responses.

Activation of murine macrophages via TLR2 and TLR4 is negatively regulated by a Lyn/PI3K module and promoted by SHIP1.

Src family kinases are involved in a plethora of aspects of cellular signaling. We demonstrate in this study that the Src family kinase Lyn negatively regulates TLR signaling in murine bone marrow-derived macrophages (BMM Phis) and in vivo. LPS-stimulated Lyn(-/-) BMM Phis produced significantly more IL-6, TNF-alpha, and IFN-alpha/beta compared with wild type (WT) BMM Phis, suggesting that Lyn is able to control both MyD88- and TRIF-dependent signaling pathways downstream of TLR4. CD14 was not involved in this type of regulation. Moreover, Lyn attenuated proinflammatory cytokine production in BMM Phis in response to the TLR2 ligand FSL-1, but not to ligands for TLR3 (dsRNA) or TLR9 (CpG 1668). In agreement with these in vitro experiments, Lyn-deficient mice produced higher amounts of proinflammatory cytokines than did WT mice after i. v. injection of LPS or FSL-1. Although Lyn clearly acted as a negative regulator downstream of TLR4 and TLR2, it did not, different from what was proposed previously, prevent the induction of LPS tolerance. Stimulation with a low dose of LPS resulted in reduced production of proinflammatory cytokines after subsequent stimulation with a high dose of LPS in both WT and Lyn(-/-) BMM Phis, as well as in vivo. Mechanistically, Lyn interacted with PI3K; in correlation, PI3K inhibition resulted in increased LPS-triggered cytokine production. In this line, SHIP1(-/-) BMM Phis, exerting enhanced PI3K-pathway activation, produced fewer cytokines than did WT BMM Phis. The data suggest that the Lyn-mediated negative regulation of TLR signaling proceeds, at least in part, via PI3K.

Bruton's tyrosine kinase is dispensable for the Toll-like receptor-mediated activation of mast cells.

Bruton's tyrosine kinase (Btk) represents an important signaling element downstream of ITAM-containing receptors, e.g. FcepsilonR1 and BCR. Btk is part of the calcium signalosome and thus, critically involved in intracellular calcium mobilization. Loss of Btk or expression of mutant forms results in severe disease phenotypes, X-linked agammaglobulinemia (XLA) and Xid in humans and mice, respectively. Previously, roles for Btk in TLR-mediated signal transduction have been found in monocytes/macrophages. In the present study we show that Btk deficiency moderately enhances or has no influence on the LPS- or lipopeptide-induced secretion of IL-6 and TNF-alpha from murine bone marrow-derived mast cells (BMMCs). Furthermore, activation of p38 kinase, which is required for cytokine production, is comparable in WT and Btk-/- BMMCs. Moreover, stability of the adaptor protein Mal as well as LPS-induced H(2)O(2) production does not vary between WT and Btk-/- cells. Interestingly, PKC-beta deficiency, which results in a Xid-like phenotype as well, has also no negative effect on LPS-induced cytokine secretion, suggesting that proteins of the calcium signalosome are not involved in TLR-mediated BMMC activation. In conclusion, the study reveals that Btk is dispensable for TLR signaling and function in murine BMMCs.

Key role of splenic myeloid DCs in the IFN-alphabeta response to adenoviruses in vivo.

The early systemic production of interferon (IFN)-alphabeta is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-alphabeta response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-alphabeta mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-alphabeta production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-alphabeta response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-alphabeta and IL-6 in vivo by distinct pathways and confirm that IFN-alphabeta positively regulates the IL-6 response. Finally, by measuring TNF-alpha responses to LPS in Ad-infected wild type and IFN-alphabetaR(-/-) mice, we show that IFN-alphabeta is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-alphabeta response, which is responsible for the bulk of IFN-alphabeta production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-alphabeta induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease.

Lipopolysaccharide sensing an important factor in the innate immune response to Gram-negative bacterial infections: benefits and hazards of LPS hypersensitivity.

In this review, we summarize our investigations concerning the differential importance of CD14 and LBP in toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2)-mediated signaling by smooth and rough-form lipopolysaccharide (LPS) chemotypes and include the results obtained in studies with murine and human TLR4-transgenic mice. Furthermore, we present more recent data on the mechanisms involved in the induction of LPS hypersensitivity by bacterial and viral infections and on the reactivity of the hypersensitive host to non-LPS microbial ligands and endogenous mediators. Finally, the effects of pre-existing hypersensitivity on the course and outcome of a super-infection with Salmonella typhimurium or Listeria monocytogenes are summarized.

R-form LPS, the master key to the activation ofTLR4/MD-2-positive cells.

Lipopolysaccharide (endotoxin, LPS) is a major recognition marker for the detection of gram-negative bacteria by the host and a powerful initiator of the inflammatory response to infection. Using S- and R-form LPS from wild-type and R-mutants of Salmonella and E. coli, we show that R-form LPS readily activates mouse cells expressing the signaling receptor Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD-2), while the S-form requires further the help of the LPS-binding proteins CD14 and LBP, which limits its activating capacity. Therefore, the R-form LPS under physiological conditions recruits a larger spectrum of cells in endotoxic reactions than S-form LPS. We also show that soluble CD14 at high concentrations enables CD14-negative cells to respond to S-form LPS. The presented in vitro data are corroborated by an in vivo study measuring TNF-alpha levels in response to injection of R- and S-form LPS in mice. Since the R-form LPS constitutes ubiquitously part of the total LPS present in all wild-type bacteria its contribution to the innate immune response and pathophysiology of infection is much higher than anticipated during the last half century.

CD14 is required for MyD88-independent LPS signaling.

The recessive mutation 'Heedless' (hdl) was detected in third-generation N-ethyl-N-nitrosourea-mutated mice that showed defective responses to microbial inducers. Macrophages from Heedless homozygotes signaled by the MyD88-dependent pathway in response to rough lipopolysaccharide (LPS) and lipid A, but not in response to smooth LPS. In addition, the Heedless mutation prevented TRAM-TRIF-dependent signaling in response to all LPS chemotypes. Heedless also abolished macrophage responses to vesicular stomatitis virus and substantially inhibited responses to specific ligands for the Toll-like receptor 2 (TLR2)-TLR6 heterodimer. The Heedless phenotype was positionally ascribed to a premature stop codon in Cd14. Our data suggest that the TLR4-MD-2 complex distinguishes LPS chemotypes, but CD14 nullifies this distinction. Thus, the TLR4-MD-2 complex receptor can function in two separate modes: one in which full signaling occurs and one limited to MyD88-dependent signaling.