Anja A. Kühl

IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases

B lymphocytes have critical roles as positive and negative regulators of immunity. Their inhibitory function has been associated primarily with interleukin 10 (IL-10) because B-cell-derived IL-10 can protect against autoimmune disease and increase susceptibility to pathogens. Here we identify IL-35-producing B cells as key players in the negative regulation of immunity. Mice in which only B cells did not express IL-35 lost their ability to recover from the T-cell-mediated demyelinating autoimmune disease experimental autoimmune encephalomyelitis (EAE). In contrast, these mice displayed a markedly improved resistance to infection with the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium as shown by their superior containment of the bacterial growth and their prolonged survival after primary infection, and upon secondary challenge, compared to control mice. The increased immunity found in mice lacking IL-35 production by B cells was associated with a higher activation of macrophages and inflammatory T cells, as well as an increased function of B cells as antigen-presenting cells (APCs). During Salmonella infection, IL-35- and IL-10-producing B cells corresponded to two largely distinct sets of surface-IgM+CD138hiTACI+CXCR4+CD1dintTim1int plasma cells expressing the transcription factor Blimp1 (also known as Prdm1). During EAE, CD138+ plasma cells were also the main source of B-cell-derived IL-35 and IL-10. Collectively, our data show the importance of IL-35-producing B cells in regulation of immunity and highlight IL-35 production by B cells as a potential therapeutic target for autoimmune and infectious diseases. This study reveals the central role of activated B cells, particularly plasma cells, and their production of cytokines in the regulation of immune responses in health and disease.

The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes

After being activated by antigen, helper T lymphocytes switch from a resting state to clonal expansion. This switch requires inactivation of the transcription factor Foxo1, a suppressor of proliferation expressed in resting helper T lymphocytes. In the early antigen-dependent phase of expansion, Foxo1 is inactivated by antigen receptor–mediated post-translational modifications. Here we show that in the late phase of expansion, Foxo1 was no longer post-translationally regulated but was inhibited post-transcriptionally by the interleukin 2 (IL-2)-induced microRNA miR-182. Specific inhibition of miR-182 in helper T lymphocytes limited their population expansion in vitro and in vivo. Our results demonstrate a central role for miR-182 in the physiological regulation of IL-2-driven helper T cell–mediated immune responses and open new therapeutic possibilities.

Anti-Inflammatory Effects of Resveratrol, Curcumin and Simvastatin in Acute Small Intestinal Inflammation

Background The health beneficial effects of Resveratrol, Curcumin and Simvastatin have been demonstrated in various experimental models of inflammation. We investigated the potential anti-inflammatory and immunomodulatory mechanisms of the above mentioned compounds in a murine model of hyper-acute Th1-type ileitis following peroral infection with Toxoplasma gondii. Methodology/Principal Findings Here we show that after peroral administration of Resveratrol, Curcumin or Simvastatin, mice were protected from ileitis development and survived the acute phase of inflammation whereas all Placebo treated controls died. In particular, Resveratrol treatment resulted in longer-term survival. Resveratrol, Curcumin or Simvastatin treated animals displayed significantly increased numbers of regulatory T cells and augmented intestinal epithelial cell proliferation/regeneration in the ileum mucosa compared to placebo control animals. In contrast, mucosal T lymphocyte and neutrophilic granulocyte numbers in treated mice were reduced. In addition, levels of the anti-inflammatory cytokine IL-10 in ileum, mesenteric lymph nodes and spleen were increased whereas pro-inflammatory cytokine expression (IL-23p19, IFN-γ, TNF-α, IL-6, MCP-1) was found to be significantly lower in the ileum of treated animals as compared to Placebo controls. Furthermore, treated animals displayed not only fewer pro-inflammatory enterobacteria and enterococci but also higher anti-inflammatory lactobacilli and bifidobacteria loads. Most importantly, treatment with all three compounds preserved intestinal barrier functions as indicated by reduced bacterial translocation rates into spleen, liver, kidney and blood. Conclusion/Significance Oral treatment with Resveratrol, Curcumin or Simvastatin ameliorates acute small intestinal inflammation by down-regulating Th1-type immune responses and prevents bacterial translocation by maintaining gut barrier function. These findings provide novel and potential prophylaxis and treatment options of patients with inflammatory bowel diseases.

Novel Murine Infection Models Provide Deep Insights into the “Ménage à Trois” of Campylobacter jejuni, Microbiota and Host Innate Immunity

Background Although Campylobacter jejuni-infections have a high prevalence worldwide and represent a significant socioeconomic burden, it is still not well understood how C. jejuni causes intestinal inflammation. Detailed investigation of C. jejuni-mediated intestinal immunopathology is hampered by the lack of appropriate vertebrate models. In particular, mice display colonization resistance against this pathogen. Methodology/Principal Findings To overcome these limitations we developed a novel C. jejuni-infection model using gnotobiotic mice in which the intestinal flora was eradicated by antibiotic treatment. These animals could then be permanently associated with a complete human (hfa) or murine (mfa) microbiota. After peroral infection C. jejuni colonized the gastrointestinal tract of gnotobiotic and hfa mice for six weeks, whereas mfa mice cleared the pathogen within two days. Strikingly, stable C. jejuni colonization was accompanied by a pro-inflammatory immune response indicated by increased numbers of T- and B-lymphocytes, regulatory T-cells, neutrophils and apoptotic cells, as well as increased concentrations of TNF-α, IL-6, and MCP-1 in the colon mucosa of hfa mice. Analysis of MyD88−/−, TRIF−/−, TLR4−/−, and TLR9−/− mice revealed that TLR4- and TLR9-signaling was essential for immunopathology following C. jejuni-infection. Interestingly, C. jejuni-mutant strains deficient in formic acid metabolism and perception induced less intestinal immunopathology compared to the parental strain infection. In summary, the murine gut flora is essential for colonization resistance against C. jejuni and can be overcome by reconstitution of gnotobiotic mice with human flora. Detection of C. jejuni-LPS and -CpG-DNA by host TLR4 and TLR9, respectively, plays a key role in immunopathology. Finally, the host immune response is tightly coupled to bacterial formic acid metabolism and invasion fitness. Conclusion/Significance We conclude that gnotobiotic and “humanized” mice represent excellent novel C. jejuni-infection and -inflammation models and provide deep insights into the immunological and molecular interplays between C. jejuni, microbiota and innate immunity in human campylobacteriosis.

The proteasome inhibitior bortezomib depletes plasma cells and ameliorates clinical manifestations of refractory systemic lupus erythematosus

Objectives To investigate whether bortezomib, a proteasome inhibitor approved for treatment of multiple myeloma, induces clinically relevant plasma cell (PC) depletion in patients with active, refractory systemic lupus erythematosus (SLE). Methods Twelve patients received a median of two (range 1–4) 21-day cycles of intravenous bortezomib (1.3 mg/m2) with the coadministration of dexamethasone (20 mg) for active SLE. Disease activity was assessed using the SLEDAI-2K score. Serum concentrations of anti–double-stranded DNA (anti-dsDNA) and vaccine-induced protective antibodies were monitored. Flow cytometry was performed to analyse peripheral blood B-cells, PCs and Siglec-1 expression on monocytes as surrogate marker for type-I interferon (IFN) activity. Results Upon proteasome inhibition, disease activity significantly declined and remained stable for 6 months on maintenance therapies. Nineteen treatment-emergent adverse events occurred and, although mostly mild to moderate, resulted in treatment discontinuation in seven patients. Serum antibody levels significantly declined, with greater reductions in anti-dsDNA (∼60%) than vaccine-induced protective antibody titres (∼30%). Bortezomib significantly reduced the numbers of peripheral blood and bone marrow PCs (∼50%), but their numbers increased between cycles. Siglec-1 expression on monocytes significantly declined. Conclusions These findings identify proteasome inhibitors as a putative therapeutic option for patients with refractory SLE by targeting PCs and type-I IFN activity, but our results must be confirmed in controlled trials.

Targeting the proteasome: partial inhibition of the proteasome by bortezomib or deletion of the immunosubunit LMP7 attenuates experimental colitis

Background and aims Inflammatory bowel disease (IBD), comprising Crohn´s disease and ulcerative colitis, is characterised by chronic relapsing inflammation of the gut. Increased proteasome activity, associated with the expression of immunoproteasomes, was found to enhance proinflammatory signalling and thus promotes inflammation in patients with IBD. The aim of this study was to explore whether modulation of the proteasomal activity is a suitable therapeutic approach to limit inflammation in colitis. Methods This concept was assessed in two different experimental set-ups. Development of dextran sodium sulfate (DSS)-induced colitis was tested (1) in lmp7−/− mice lacking the immunoproteasome subunit LMP7 and (2) in wild-type (WT) mice treated with the proteasome inhibitor bortezomib. Results Compared with WT mice, lmp7−/− mice develop significantly attenuated colitis due to reduced nuclear factor-κB (NF-κB) signalling in the absence of LMP7. Further, treatment with bortezomib revealed dose-dependent amelioration of DSS-induced inflammation. In both approaches modulation of the proteasome activity limited the secretion of proinflammatory cytokines and chemokines. Consequently, infiltration of the colon by neutrophils and expansion of inflammatory T helper 1 (Th1) and Th17 T cells was diminished and thus prevented excessive tissue damage. Conclusions It was demonstrated that modulation of the proteasome activity is effective in attenuating experimental colitis. The results reveal that reduction of the proteasome activity either by partial inhibition with bortezomib or by specifically targeting the immunoproteasome subunit LMP7 is a suitable treatment of intestinal inflammation.

Animal Models of Inflammatory Bowel Disease: An Overview

Inflammatory bowel disease (IBD) research has been performed in human in vitro studies and in in vivo studies using appropriate animal models. Such animal models allow both the examination of inflammatory processes (both early and late events) as well as the evaluation of new therapeutic modalities. Since the first description of the immune complex colitis in rabbits in 1961, overall 63 models have been described, most of which within the last decade. These IBD animal models can be divided into 5 different categories: (1) antigen-induced colitis and colitis induced by microbials; (2) other inducible forms of colitis (chemical, immunological, and physical); (3) genetic colitis models (transgenic and knock-out models); (4) adoptive transfer models, and (5) spontaneous colitis models. In spite of the high overall number of models, none of them is the ‘perfect’ model and therefore numerous aspects need to be considered when choosing one model for a particular study. Importantly, most clinical aspects (e.g. extraintestinal manifestations or fistula) have recently been described in one or the other model allowing further studies with relevance for almost all aspects of IBD. So far, IBD animal models have taught us important lessons, e.g. the requirement of T-helper cells in most models, the need of a particular genetic background, and the role of the flora in the initiation of IBD. It is expected that our understanding of IBD will further increase in a number of additional areas using animal models, e.g. exploring the role of the innate immune system in IBD.

Intestinal Microbiota Shifts towards Elevated Commensal Escherichia coli Loads Abrogate Colonization Resistance against Campylobacter jejuni in Mice

Background The zoonotic pathogen Campylobacter jejuni is a leading cause of bacterial foodborne enterocolitis in humans worldwide. The understanding of immunopathology underlying human campylobacteriosis is hampered by the fact that mice display strong colonization resistance against the pathogen due to their host specific gut microbiota composition. Methodology/Principal Findings Since the microbiota composition changes significantly during intestinal inflammation we dissected factors contributing to colonization resistance against C. jejuni in murine ileitis, colitis and in infant mice. In contrast to healthy animals C. jejuni could stably colonize mice suffering from intestinal inflammation. Strikingly, in mice with Toxoplasma gondii-induced acute ileitis, C. jejuni disseminated to mesenteric lymphnodes, spleen, liver, kidney, and blood. In infant mice C. jejuni infection induced enterocolitis. Mice suffering from intestinal inflammation and C. jejuni susceptible infant mice displayed characteristical microbiota shifts dominated by increased numbers of commensal Escherichia coli. To further dissect the pivotal role of those distinct microbiota shifts in abrogating colonization resistance, we investigated C. jejuni infection in healthy adult mice in which the microbiota was artificially modified by feeding live commensal E. coli. Strikingly, in animals harboring supra-physiological intestinal E. coli loads, colonization resistance was significantly diminished and C. jejuni infection induced enterocolitis mimicking key features of human campylobacteriosis. Conclusion/Significance Murine colonization resistance against C. jejuni is abrogated by changes in the microbiota composition towards elevated E. coli loads during intestinal inflammation as well as in infant mice. Intestinal inflammation and microbiota shifts thus represent potential risk factors for C. jejuni infection. Corresponding interplays between C. jejuni and microbiota might occur in human campylobacteriosis. Murine models introduced here mimick key features of human campylobacteriosis and allow for further analysis of immunological and molecular mechanisms of C. jejuni – host interactions.

Vascular Receptor Autoantibodies in Pulmonary Arterial Hypertension Associated with Systemic Sclerosis

RATIONALE Systemic sclerosis (SSc)-associated pulmonary arterial hypertension (PAH) portends worse outcome than other forms of PAH. Vasoconstrictive and vascular remodeling actions of endothelin (ET) 1 and angiotensin (Ang) II via endothelin receptor type A (ETAR) and Ang receptor type-1 (AT1R) activation are implicated in PAH pathogenesis. OBJECTIVES We hypothesized that stimulating autoantibodies (Abs) targeting and activating AT1R and ETAR may contribute to SSc-PAH pathogenesis, and tested their functional and biomarker relevance. METHODS Anti-AT1R and -ETAR Abs were detected by ELISA in different cohorts of patients and tested in vitro and in an animal model for their pathophysiological effects. MEASUREMENTS AND MAIN RESULTS The Abs were significantly higher and more prevalent in patients with SSc-PAH (n = 81) and connective tissue disease-associated PAH (n = 110) compared with other forms of PAH/pulmonary hypertension (n = 106). High anti-AT1R and anti-ETAR Abs predicted development of SSc-PAH and SSc-PAH-related mortality in a prospective analysis. Both Abs increased endothelial cytosolic Ca(2+) concentrations in isolated perfused rat lungs, which could be blocked by respective specific receptor antagonists. Ab-mediated stimulation of intralobar pulmonary rat artery ring segments increased vasoconstrictive responses to Ang II and ET-1, and implicated cross-talk between both pathways demonstrated by reciprocal blockade with respective antagonists. Transfer of SSc-IgG containing both autoantibodies into healthy C57BL/6J mice led to more abundant vascular and airway α-smooth muscle actin expression and inflammatory pulmonary vasculopathy. CONCLUSIONS Anti-AT1R and -ETAR Abs are more frequent in SSc-PAH/connective tissue disease-PAH compared with other forms of pulmonary hypertension, and serve as predictive and prognostic biomarkers in SSc-PAH. Both antibodies may contribute to SSc-PAH via increased vascular endothelial reactivity and induction of pulmonary vasculopathy.

Stable T-bet(+)GATA-3(+) Th1/Th2 hybrid cells arise in vivo, can develop directly from naive precursors, and limit immunopathologic inflammation.

The stable lineage commitment of naïve T helper cells to a hybrid Th1/2 phenotype reveals the cell-intrinsic reconciliation of two opposing T cell differentiation programs and provides a self-limiting mechanism to dampen immunopathology.