Kotlarz Group

We are aiming to decode disease signatures of Pediatric IBD using a system biology-driven approach

The mission of our research group is to explore the molecular causes in children with very early onset inflammatory bowel disease (VEO-IBD), a life-threatening condition. In particular, our laboratory focuses on decoding genetic and immune signatures of VEO-IBD by employing omics-based technologies and advanced preclinical models. We propose that our studies will lead to new insights into disease pathogenesis, diagnosis, and treatment for children with this intractable disease. Specifically, we investigate:

Genetic signatures in pediatric inflammatory bowel disease
Transcriptional networks in pediatric inflammatory bowel disease
Mucosal Immunology
Preclinical Models of inflammatory bowel disease

Inflammatory bowel disease is a multifactorial disorder of the digestive tract triggered by environmental factors, immune dysfunctions, defective epithelial barrier function, and imbalances of the microbial flora in genetically susceptible individuals.

About 20% of patients with IBD are diagnosed during childhood and adolescents. Children with very early onset inflammatory bowel diseases (VEO-IBD, age of onset <6 years) often show severe and life-threatening conditions refractory to conventional treatment.

Paradigmatic studies by our laboratory have shown that VEO-IBD can be caused by monogenic IL-10R defects (Glocker et al, New Engl J Med 2009). Based on the knowledge of the underlying molecular etiology, IL-10R-deficient patients could be treated with allogeneic hematopoietic stem cell transplantation, an innovative therapeutic approach for defined patients with IBD (Kotlarz et al, Gastroenterology 2012). This prime example of translational research demonstrated the importance of genetic diagnostics for the clinical management of VEO-IBD patients and highlighted that rare variants of IBD represent exquisite models to identify key molecular factors controlling intestinal homeostasis.

The overall goal of our laboratory is to explore the molecular pathomechanisms of VEO-IBD in order to develop novel diagnostic tools and therapies for children with intractable colitis.

In collaboration with our partners in Boston (Dr. Scott Snapper) and Toronto (Dr. Aleixo Muise), we have established an international VEO-IBD consortium that will be supported by world experts in the field of immunology, genetics, genetic engineering, gastroenterology, intestinal stem cell biology, microbiomics and bioinformatics.

To elucidate novel genetic signatures of VEO-IBD, we have established collaborations to more than 150 international clinical institutes and systematically screened one of the largest international cohorts of VEO-IBD by employing state-of-the-art next-generation sequencing. As proof-of-principle, our laboratory has characterized first VEO-IBD patients with TGFB1 (Kotlarz et al., Nat Genet 2018), CASP8 (Lehle et al., Gastroenterology 2019), and RIPK1 (Li et al., PNAS 2019) deficiency. Our computational analysis has also unraveled several novel candidate genes that might be implicated in the pathogenesis of VEO-IBD.

We will analyze the molecular pathomechanisms of newly identified sequence variants by employing various experimental models (patient samples, heterologous model systems, mouse models) and state-of-the-art molecular and cell biological technologies.

Despite advances in genome-wide sequencing, >75 % of VEO-IBD patients lack definitive genetic diagnosis. In addition, the disease mechanisms of most known genetic entities of VEO-IBD remain largely elusive and need to be further defined in order to develop personalized therapies.

Intestinal inflammation is likely driven by alterations in tissue composition and cell-intrinsic cellular programs. In the past, transcriptomics studies have been hampered by analysis of bulk samples across entire tissues. We postulate that innovative single cell genomic analysis will allow comprehensive mapping of known and previously uncharacterized epithelial, stromal, and immune cell types as well as transcriptional disease states in complex intestinal tissues of VEO-IBD patients. Unbiased and multidimensional single cell transcriptomic data will facilitate the discovery of dysregulated inflammatory expression programs, thus providing critical insights into disease etiology and highlighting new therapeutic interventions.

To dissect the molecular pathomechanisms of VEO-IBD, our laboratory has established an experimental platform to (i) conduct state-of-the-art assays on primary patients’ cells, (ii) patient-derived induced pluripotent stem cells and intestinal 3D mini guts (intestinal organoids), (iii) generate heterologous cellular models by genetic engineering (lentiviral gene transfer, CRISPR/Cas9), and (iv) analyze patient-derived humanized mouse models by cutting-edge cell biology, biochemical and immunological assays.

The overall goal of our research is to advance the understanding of key factors in IBD pathogenesis by employing omics-based technologies and preclinical disease models. This knowledge provides the groundwork for the development of personalized therapies in order to improve life quality for children suffering from life-threatening diseases.

Kotlarz D

Identification of the γδ IEL-secreted Paneth Cell Protective Factor API5 as a Promising Therapeutic Target for ATG16L1-associated Crohn's Disease

Li Y, Yu Z, Schenk M, Lagovsky I, Illig D, Walz C, Rohlfs M, Conca R, Muise AM, Snapper SB, Uhlig HH, Garty BZ, Klein C, Kotlarz D

Human MD2 deficiency-an inborn error of immunity with pleiotropic features Background: Toll-like receptors (TLRs) are important pattern recognition receptors that sense microbes and control host defense. Myeloid differentiation protein 2 (MD2) is the indispensable coreceptor for TLR4, facilitating the binding to the gram-negative bacterial cell wall component LPS and activation of downstream signaling.

Objective: We sought to provide phenotypic and mechanistic insights into human MD2 deficiency.

Methods: To elucidate the genetic cause in a patient with very early onset inflammatory bowel disease, we performed whole-exome sequencing and studied the functional consequences of the identified mutation in LY96 (encoding for MD2) in genetically engineered induced pluripotent stem cell-derived macrophages with knockout of MD2 or knockin of the patient-specific mutation, including TLR4-mediated signaling, cytokine production, and bacterial handling.

Results: Whole-exome sequencing identified a homozygous in-frame deletion in the LY96 gene (c.347_349delCAA; p.Thr116del) in a patient with very early onset inflammatory bowel disease and a sibling presenting with pneumonia and otitis media. Induced pluripotent stem cell-derived macrophages with knockout of MD2 or expression of the Thr116del mutation showed impaired activation of nuclear factor kappa B and mitogen-activated protein kinase signaling as well as TLR4 endocytosis on challenge with LPS or bacteria. In addition, MD2-deficient macrophages showed decreased cytokine expression (eg, IL-6, TNF, and IL-10) in response to LPS or gram-negative but not gram-positive bacteria.

Conclusions: Human MD2 deficiency causes defective TLR4 signaling in response to LPS or gram-negative bacteria. The clinical manifestations and expressivity might be variable due to unknown secondary risk factors. Because TLR4 represents a therapeutic target for multiple inflammatory conditions, our study may provide insights into potential side effects of pharmacological TLR4 targeting.

Ghalandary M, Li Y, Fröhlich T, Magg T, Liu Y, Rohlfs M, Hollizeck S, Conca R, Schwerd T, Uhlig HH, Bufler P, Koletzko S, Muise AM, Snapper SB, Hauck F, Klein C, Kotlarz D

Valosin-containing protein-regulated endoplasmic reticulum stress causes NOD2-dependent inflammatory responses NOD2 polymorphisms may affect sensing of the bacterial muramyl dipeptide (MDP) and trigger perturbed inflammatory responses. Genetic screening of a patient with immunodeficiency and enteropathy revealed a rare homozygous missense mutation in the first CARD domain of NOD2 (ENST00000300589; c.160G > A, p.E54K). Biochemical assays confirmed impaired NOD2-dependent signaling and proinflammatory cytokine production in patient's cells and heterologous cellular models with overexpression of the NOD2 mutant. Immunoprecipitation-coupled mass spectrometry unveiled the ATPase valosin-containing protein (VCP) as novel interaction partner of wildtype NOD2, while the binding to the NOD2 variant p.E54K was abrogated. Knockdown of VCP in coloncarcinoma cells led to impaired NF-κB activity and IL8 expression upon MDP stimulation. In contrast, tunicamycin-induced ER stress resulted in increased IL8, CXCL1, and CXCL2 production in cells with knockdown of VCP, while enhanced expression of these proinflammatory molecules was abolished upon knockout of NOD2. Taken together, these data suggest that VCP-mediated inflammatory responses upon ER stress are NOD2-dependent.

Khoshnevisan R, Anderson M, Babcock S, Anderson S, Illig D, Marquardt B, Sherkat R, Schröder K, Moll F, Hollizeck S, Rohlfs M, Walz C, Adibi P, Rezaei A, Andalib A, Koletzko S, Muise AM, Snapper SB, Klein C, Thiagarajah JR, Kotlarz D (2020). Inflamm Bowel Dis. 17.

NOX1 Regulates Collective and Planktonic Cell Migration: Insights From Patients With Pediatric-Onset IBD and NOX1 Deficiency. Genetic defects of pediatric-onset inflammatory bowel disease (IBD) provide critical insights into molecular factors controlling intestinal homeostasis. NOX1 has been recently recognized as a major source of reactive oxygen species (ROS) in human colonic epithelial cells. Here we assessed the functional consequences of human NOX1 deficiency with respect to wound healing and epithelial migration by studying pediatric IBD patients presenting with a stop-gain mutation in NOX1.

Li Y, Klein C, Kotzlarz D

Dysregulation of Cell Death in Human Chronic Inflammation Inflammation is a fundamental biological process mediating host defense and wound healing during infections and tissue injury. Perpetuated and excessive inflammation may cause autoinflammation, autoimmunity, degenerative disorders, allergies, and malignancies. Multimodal signaling by tumor necrosis factor receptor 1 (TNFR1) plays a crucial role in determining the transition between inflammation, cell survival, and programmed cell death. Targeting TNF signaling has been proven as an effective therapeutic in several immune-related disorders. Mouse studies have provided critical mechanistic insights into TNFR1 signaling and its potential role in a broad spectrum of diseases. The characterization of patients with monogenic primary immunodeficiencies (PIDs) has highlighted the importance of TNFR1 signaling in human disease. In particular, patients with PIDs have revealed paradoxical connections between immunodeficiency, chronic inflammation, and dysregulated cell death. Importantly, studies on PIDs may help to predict beneficial effects and side-effects of therapeutic targeting of TNFR1 signaling.

Li Y, Fuhrer M, Bahrami E, Socha P, Klaudel-Dreszler M, Bouzidi A, Liu Y, Lehle AS, Magg T, Hollizeck S, Rohlfs M, Conca R, Field M, Warner N, Mordechai S, Shteyer E, Turner D, Boukari R, Belbouab R, Walz C, Gaidt MM, Hornung V, Baumann B, Pannicke U, Al Idrissi E, Ali Alghamdi H, Sepulveda FE, Gil M, de Saint Basile G, Honig M, Koletzko S, Muise AM, Snapper SB, Schwarz K, Klein C, and Kotlarz D

Human RIPK1 deficiency causes combined immunodeficiency and inflammatory bowel diseases Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a critical regulator of cell death and inflammation, but its relevance for human disease pathogenesis remains elusive. Studies of monogenic disorders might provide critical insights into disease mechanisms and therapeutic targeting of RIPK1 for common diseases. Here, we report on eight patients from six unrelated pedigrees with biallelic loss-of-function mutations in RIPK1 presenting with primary immunodeficiency and/or intestinal inflammation. Mutations in RIPK1 were associated with reduced NF-κB activity, defective differentiation of T and B cells, increased inflammasome activity, and impaired response to TNFR1-mediated cell death in intestinal epithelial cells. The characterization of RIPK1-deficient patients highlights the essential role of RIPK1 in controlling human immune and intestinal homeostasis, and might have critical implications for therapies targeting RIPK1.

Lehle AS, Farin HF, Marquardt B, Michels BE, Magg T, Li Y, Liu Y, Ghalandary M, Lammens K, Hollizeck S, Rohlfs M, Hauck F, Conca R, Walz C, Weiss B, Lev A, Simon AJ, Gross O, Gaidt MM, Hornung V, Clevers H, Yazbeck N, Hanna-Wakim R, Shouval DS, Warner N, Somech R, Muise AM, Snapper SB, Bufler P, Koletzko S, Klein C, and Kotlarz D

Intestinal Inflammation and Dysregulated Immunity in Patients With Inherited Caspase-8 Deficiency

Kotlarz D, Marquardt B, Baroy T, Lee WS, Konnikova L, Hollizeck S, Magg T, Lehle AS, Walz C, Borggraefe I, Hauck F, Bufler P, Conca R, Wall SM, Schumacher EM, Misceo D, Frengen E, Bentsen BS, Uhlig HH, Hopfner KP, Muise AM, Snapper SB, Stromme P, and Klein C

Human TGF-beta1 deficiency causes severe inflammatory bowel disease and encephalopathy Transforming growth factor (TGF)-β1 (encoded by TGFB1) is the prototypic member of the TGF-β family of 33 proteins that orchestrate embryogenesis, development and tissue homeostasis1,2. Following its discovery3, enormous interest and numerous controversies have emerged about the role of TGF-β in coordinating the balance of pro- and anti-oncogenic properties4,5, pro- and anti-inflammatory effects6, or pro- and anti-fibrinogenic characteristics7. Here we describe three individuals from two pedigrees with biallelic loss-of-function mutations in the TGFB1 gene who presented with severe infantile inflammatory bowel disease (IBD) and central nervous system (CNS) disease associated with epilepsy, brain atrophy and posterior leukoencephalopathy. The proteins encoded by the mutated TGFB1 alleles were characterized by impaired secretion, function or stability of the TGF-β1–LAP complex, which is suggestive of perturbed bioavailability of TGF-β1. Our study shows that TGF-β1 has a critical and nonredundant role in the development and homeostasis of intestinal immunity and the CNS in humans.

Kotlarz D, Zietara N, Uzel G, Weidemann T, Braun CJ, Diestelhorst J, Krawitz PM, Robinson PN, Hecht J, Puchalka J, Gertz EM, Schaffer AA, Lawrence MG, Kardava L, Pfeifer D, Baumann U, Pfister ED, Hanson EP, Schambach A, Jacobs R, Kreipe H, Moir S, Milner JD, Schwille P, Mundlos S, and Klein C

Loss-of-function mutations in the IL-21 receptor gene cause a primary immunodeficiency syndrome Primary immunodeficiencies (PIDs) represent exquisite models for studying mechanisms of human host defense. In this study, we report on two unrelated kindreds, with two patients each, who had cryptosporidial infections associated with chronic cholangitis and liver disease. Using exome and candidate gene sequencing, we identified two distinct homozygous loss-of-function mutations in the interleukin-21 receptor gene (IL21R; c.G602T, p.Arg201Leu and c.240_245delCTGCCA, p.C81_H82del). The IL-21RArg201Leu mutation causes aberrant trafficking of the IL-21R to the plasma membrane, abrogates IL-21 ligand binding, and leads to defective phosphorylation of signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5. We observed impaired IL-21–induced proliferation and immunoglobulin class-switching in B cells, cytokine production in T cells, and NK cell cytotoxicity. Our study indicates that human IL-21R deficiency causes an immunodeficiency and highlights the need for early diagnosis and allogeneic hematopoietic stem cell transplantation in affected children.

Kotlarz D, Beier R, Murugan D, Diestelhorst J, Jensen O, Boztug K, Pfeifer D, Kreipe H, Pfister ED, Baumann U, Puchalka J, Bohne J, Egritas O, Dalgic B, Kolho KL, Sauerbrey A, Buderus S, Gungor T, Enninger A, Koda YK, Guariso G, Weiss B, Corbacioglu S, Socha P, Uslu N, Metin A, Wahbeh GT, Husain K, Ramadan D, Al-Herz W, Grimbacher B, Sauer M, Sykora KW, Koletzko S, and Klein C

Loss of interleukin-10 signaling and infantile inflammatory bowel disease: implications for diagnosis and therapy Homozygous loss of function mutations in interleukin-10 (IL10) and interleukin-10 receptors (IL10R) cause severe infantile (very early onset) inflammatory bowel disease (IBD). Allogeneic hematopoietic stem cell transplantation (HSCT) was reported to induce sustained remission in 1 patient with IL-10R deficiency. We investigated heterogeneity among patients with very early onset IBD, its mechanisms, and the use of allogeneic HSCT to treat this disorder

Glocker EO, Kotlarz D, Boztug K, Gertz EM, Schaffer AA, Noyan F, Perro M, Diestelhorst J, Allroth A, Murugan D, Hatscher N, Pfeifer D, Sykora KW, Sauer M, Kreipe H, Lacher M, Nustede R, Woellner C, Baumann U, Salzer U, Koletzko S, Shah N, Segal AW, Sauerbrey A, Buderus S, Snapper SB, Grimbacher B, and Klein C

Inflammatory bowel disease and mutations affecting the interleukin-10 receptor. The molecular cause of inflammatory bowel disease is largely unknown.

Kotlarz Group

Scientific Focus

Our Projects

Research Background

Unravelling genetic signatures of pediatric IBD

Decoding transcriptional networks of pediatric IBD

Application of preclinical models of IBD

Development of personalized therapies

The Kotlarz Group

Selected Publications