Dmitry V. Kuprash

Distinct Role of Surface Lymphotoxin Expressed by B Cells in the Organization of Secondary Lymphoid Tissues

In order to definitively ascertain the functional contribution of lymphotoxin (LT) expressed by B cells, we produced mice with the LTbeta gene deleted from B cells (B-LTbeta KO mice). In contrast to systemic LTbeta deletion, in B-LTbeta KO mice only splenic microarchitecture was affected, while lymph nodes and Peyers patches (PP) were normal, except for PPs reduced size. Even though B-LTbeta KO spleens retained a small number of follicular dendritic cells (FDC) which appeared to be dependent on LTbeta produced by T cells, IgG responses to sheep red blood cells were markedly reduced. Thus, the organogenic function of B-LTbeta is almost entirely restricted to spleen, where it supports the correct lymphoid architecture that is critical for an effective humoral immune response.

p53 is a suppressor of inflammatory response in mice

Chronic inflammation is known to promote cancer, suggesting that negative regulation of inflammation is likely to be tumor suppressive. We found that p53 is a general inhibitor of inflammation that acts as an antagonist of nuclear factor κB (NFκB). We first observed striking similarities in global gene expression profiles in human prostate cancer cells LNCaP transduced with p53 inhibitory genetic element or treated with TNF, suggesting that p53 inhibits transcription of TNF‐inducible genes that are largely regulated by NFκB. Consistently, ectopically expressed p53 acts as an inhibitor of transcription of NFκB‐dependent promoters. Furthermore, suppression of inflammatory response by p53 was observed in vivo in mice by comparing wild‐type and p53 null animals at molecular (inhibition of transcription of genes encoding cytokines and chemokines, reducing accumulation of reactive oxygen species and protein oxidation products), cellular (activation of macrophages and neutrophil clearance) and organismal (high levels of metabolic markers of inflammation in tissues of p53‐deficient mice and their hypersensitivity to LPS) levels. These observations indicate that p53, acting through suppression of NFκB, plays the role of a general “buffer” of innate immune response in vivo that is well consistent with its tumor suppressor function and frequent constitutive activation of NFκB in tumors.

Nonredundant Function of Soluble LTα3 Produced by Innate Lymphoid Cells in Intestinal Homeostasis

Command and Control Innate lymphoid cells are vital for the development of gut-associated lymphoid tissues, maintenance of the epithelial barrier, and protection against intestinal microbes; their dysfunction can promote immune pathology. Immunoglobulin A (IgA) production is important for maintenance of the gut epithelial barrier and the composition of the gut microbiota. Through the generation of knockout mouse models, Kruglov et al. (p. 1243) were able to distinguish how soluble and membrane-bound lymphotoxins expressed by innate lymphoid cells in the gut specifically regulate IgA production and thereby control gut microbiota composition. Soluble lymphotoxin plays a paracrine role in controlling immunoglobulin A responses and regulating gut microbiota. Immunoglobulin A (IgA) production at mucosal surfaces contributes to protection against pathogens and controls intestinal microbiota composition. However, mechanisms regulating IgA induction are not completely defined. We show that soluble lymphotoxin α (sLTα3) produced by RORγt+ innate lymphoid cells (ILCs) controls T cell–dependent IgA induction in the lamina propria via regulation of T cell homing to the gut. By contrast, membrane-bound lymphotoxin β (LTα1β2) produced by RORγt+ ILCs is critical for T cell–independent IgA induction in the lamina propria via control of dendritic cell functions. Ablation of LTα in RORγt+ cells abrogated IgA production in the gut and altered microbiota composition. Thus, soluble and membrane-bound lymphotoxins produced by ILCs distinctly organize adaptive immune responses in the gut and control commensal microbiota composition.

Lymphotoxin Beta Receptor Signaling in Intestinal Epithelial Cells Orchestrates Innate Immune Responses against Mucosal Bacterial Infection

Epithelial cells provide the first line of defense against mucosal pathogens; however, their coordination with innate and adaptive immune cells is not well understood. Using mice with conditional gene deficiencies, we found that lymphotoxin (LT) from innate cells expressing transcription factor RORgammat, but not from adaptive T and B cells, was essential for the control of mucosal C. rodentium infection. We demonstrate that the LTbetaR signaling was required for the regulation of the early innate response against infection. Furthermore, we have revealed that LTbetaR signals in gut epithelial cells and hematopoietic-derived cells coordinate to protect the host from infection. We further determined that LTbetaR signaling in intestinal epithelial cells was required for recruitment of neutrophils to the infection site early during infection via production of CXCL1 and CXCL2 chemokines. These results support a model wherein LT from RORgammat(+) cells orchestrates the innate immune response against mucosal microbial infection.

Dissecting the role of lymphotoxin in lymphoid organs by conditional targeting

Summary:  Mice with inactivation of lymphotoxin β receptor (LTβR) system have profound defects in the development and maintenance of peripheral lymphoid organs. As surface LT is expressed by lymphocytes, natural killer cells, and lymphoid tissue‐initiating cells as well as by some other cell types, we dissected cell type‐specific LT contribution into the complex LT‐deficient phenotype by conditional gene targeting. B‐LTβ knockout (KO) mice displayed an intermediate phenotype in spleen as compared with mice with complete LTβ deficiency. In contrast, T‐LTβ KO mice displayed normal structure of the spleen. However, inactivation of LTβ in both T and B cells resulted in additional defects in the structure of the marginal zone and in the development of follicular dendritic cells in spleen. Structure of lymph nodes (LN) and Peyers patches (PP) was normal in both B‐LTβ KO and T‐ and B‐LTβ KO mice, except that PPs were of reduced size. When compared across the panel of lymphocyte‐specific LT KOs, the defects in antibody responses to T‐cell‐dependent antigens correlated with the severity of defects in spleen structure. Expression of CCL21 and CCL19 chemokines was not affected in spleen, LN and PP of B‐LTβ KO and T‐ and B‐LTβ KO mice, while CXCL13 was slightly reduced only in spleen. Collectively, our data suggest the following: (i) requirements for LT signaling to support architecture of spleen, LN and PP are different; (ii) LT complex expressed by B cells plays a major role in the maintenance of spleen structure, while surface LT expressed by T cells provides a complementary but distinct signal; and (iii) in a non‐transgenic model, expression of lymphoid tissue chemokines is only minimally dependent on the expression of surface LT complex on B and T lymphocytes.

Intracellular signals and events activated by cytokines of the tumor necrosis factor superfamily: From simple paradigms to complex mechanisms.

Tumor necrosis factor (TNF) and several related cytokines can induce opposite effects such as cell activation and proliferation or cell death. How the cell maintains the balance between these seemingly mutually exclusive pathways has long remained a mystery. TNF receptor I (TNFRI) initially emerged as a potent activator of NFkappaB and AP-1 transcription factors, while the related CD95 (Fas, Apo-1) was recognized as a prototype death receptor. Advances in research have uncovered critical molecular players in these intracellular processes. They have also revealed a much more complex picture than originally thought. Several new signaling pathways, including the alternative NFkappaB activation cascade, have been uncovered, and previously unknown modes of cross-talk between intracellular signaling molecules were revealed. It also turned out that signaling mechanisms mediated by the TNF receptor superfamily members can operate not only in the immune system but also in organ development.

Physiological functions of tumor necrosis factor and the consequences of its pathologic overexpression or blockade: mouse models.

TNF is an exciting cytokine which has helped to establish many paradigms in immunology. Although TNF itself has found only very limited use in the clinic, anti-cytokine therapy, which targets this single molecule, has enjoyed astounding success in treatment of a growing number of human diseases. However, since TNF mediates unique physiologic functions, in particular those related to host defense, TNF blockade may result in unwanted consequences. Much of our understanding about TNF intrinsic functions in the body, as well as about consequences of its overexpression and ablation, is based on studying phenotypes of various genetically engineered mice. Here we review mouse studies aimed at understanding TNF physiologic functions using transgenic and knockout models, and we discuss additional mouse models that may be helpful in the future.

Novel tumor necrosis factor‐knockout mice that lack Peyer's patches

We generated a novel tumor necrosis factor (TNF) null mutation using Cre‐loxP technology. Mice homozygous for this mutation differ from their “conventional” counterparts; in particular, they completely lack Peyers patches (PP) but retain all lymph nodes. Our analysis of these novel TNF‐knockout mice supports the previously disputed notion of the involvement of TNF‐TNFR1 signaling in PP organogenesis. Availability of TNF‐knockout strains both with and without PP enables more definitive studies concerning the roles of TNF and PP in various immune functions and disease conditions. Here, we report that systemic ablation of TNF, but not the presence of PP per se, is critical for protection against intestinal Listeria infection in mice.

Redundancy in Tumor Necrosis Factor (TNF) and Lymphotoxin (LT) Signaling In Vivo: Mice with Inactivation of the Entire TNF/LT Locus versus Single-Knockout Mice

ABSTRACT Homologous genes and gene products often have redundant physiological functions. Members of the tumor necrosis factor (TNF) family of cytokines can signal activation, proliferation, differentiation, costimulation, inhibition, or cell death, depending on the type and status of the target cell. TNF, lymphotoxin α (LTα), and LTβ form a subfamily of a larger family of TNF-related ligands with their genes being linked within a compact 12-kb cluster inside the major histocompatibility complex locus. Singly TNF-, LTα-, and LTβ-deficient mice share several phenotypic features, suggesting that TNF/LT signaling pathways may regulate overlapping sets of target genes. In order to directly address the issue of redundancy of TNF/LT signaling, we used the Cre-loxP recombination system to create mice with a deletion of the entire TNF/LT locus. Mice with a triple LTβ/TNF/LTα deficiency essentially manifest a combination of LT and TNF single-knockout phenotypes, except for microarchitecture of the spleen, where the disorder of lymphoid cell positioning and functional T- and B-cell compartmentalization is severer than that found in TNF or LT single-knockout mice. Thus, our data support the notion that TNF and LT have largely nonredundant functions in vivo.

Lymphotoxin and TNF Produced by B Cells Are Dispensable for Maintenance of the Follicle-Associated Epithelium but Are Required for Development of Lymphoid Follicles in the Peyer’s Patches

Organogenesis of Peyer‘s patches (PP), follicle-associated epithelium, and M cells is impaired in mice lacking B cells. At the same time, lymphotoxin (LT) and TNF are known to be critical for the development of PP. To directly address the function of LT and TNF expressed by B cells in the maintenance of PP structure, we studied the de novo formation of PP in B cell-deficient mice after the transfer of bone marrow from mice with targeted mutations in LT, TNF, or their combinations. We found that although the compartmentalization of T and B cell zones and development of follicular dendritic cells were affected by the lack of B cell-derived LT and TNF, the development of follicle-associated epithelium and M cells in PP was completely independent of LT/TNF production by B cells.