Anja Lechner

Immunization with a heat-killed preparation of the environmental bacterium Mycobacterium vaccae promotes stress resilience in mice

Significance The hygiene, or “old friends,” hypothesis proposes that lack of exposure to immunoregulatory microorganisms in modern urban societies is resulting in an epidemic of inflammatory disease, as well as psychiatric disorders in which chronic, low-level inflammation is a risk factor. An important determinant of immunoregulation is the microbial community occupying the host organism, collectively referred to as the microbiota. Here we show that stress disrupts the homeostatic relationship between the microbiota and the host, resulting in exaggerated inflammation. Treatment of mice with a heat-killed preparation of an immunoregulatory environmental microorganism, Mycobacterium vaccae, prevents stress-induced pathology. These data support a strategy of “reintroducing” humans to their old friends to promote optimal health and wellness. The prevalence of inflammatory diseases is increasing in modern urban societies. Inflammation increases risk of stress-related pathology; consequently, immunoregulatory or antiinflammatory approaches may protect against negative stress-related outcomes. We show that stress disrupts the homeostatic relationship between the microbiota and the host, resulting in exaggerated inflammation. Repeated immunization with a heat-killed preparation of Mycobacterium vaccae, an immunoregulatory environmental microorganism, reduced subordinate, flight, and avoiding behavioral responses to a dominant aggressor in a murine model of chronic psychosocial stress when tested 1–2 wk following the final immunization. Furthermore, immunization with M. vaccae prevented stress-induced spontaneous colitis and, in stressed mice, induced anxiolytic or fear-reducing effects as measured on the elevated plus-maze, despite stress-induced gut microbiota changes characteristic of gut infection and colitis. Immunization with M. vaccae also prevented stress-induced aggravation of colitis in a model of inflammatory bowel disease. Depletion of regulatory T cells negated protective effects of immunization with M. vaccae on stress-induced colitis and anxiety-like or fear behaviors. These data provide a framework for developing microbiome- and immunoregulation-based strategies for prevention of stress-related pathologies.

Chronic psychosocial stress promotes systemic immune activation and the development of inflammatory Th cell responses.

Recent studies indicate that chronic psychosocial stress favors the development of generalized immune dysfunction. During stressor exposure neuroendocrine factors affect numbers and functionality of leukocytes. However, the exact mechanisms leading to systemic changes in immune functions during stress are still not clear. During chronic subordinate colony housing, a model of chronic psychosocial stress, mice developed spontaneous colonic inflammation. Decreased glucocorticoid signaling, induced by a combination of adrenal insufficiency and glucocorticoid resistance, was thought to prevent tempering of local immune cells, and to promote tissue inflammation. In this study we investigated changes in the systemic immune status after chronic subordinate colony housing and analyzed potential mechanisms underlying those alterations. Analysis of T helper cell subsets in peripheral lymph nodes revealed a reduction of regulatory T cells, accompanied by increased T cell effector functions. Generalized activation of T cells was shown by elevated cytokine production upon stimulation. In addition, we observed no apparent shift towards T helper type 2 responses. It is likely, that the previously reported hypocorticism in this stress model led to a steady production of inflammatory Th1, Th2, and Th17 cytokines and obstructed the shift towards an anti-inflammatory response. In conclusion, we established chronic subordinate colony housing as a model to investigate the outcome of stress on the systemic immune status. We also provide evidence that distinct T helper cell subtypes react differentially to the suppressive effect of glucocorticoids.

Sepsis leads to a reduced antigen-specific primary antibody response

Immunosuppression, impaired cytokine production and high susceptibility to secondary infections are characteristic for septic patients, and for mice after induction of polymicrobial septic peritonitis by sublethal cecal ligation and puncture (CLP). Here, we demonstrate that CLP markedly altered subsequent B‐cell responses. Total IgG and IgM levels, as well as the memory B‐cell response, were increased in septic mice, but antigen‐specific primary antibody production was strongly impaired. We found that two days after CLP, CD11b+ splenocytes were activated as demonstrated by the increased expression of activation markers, expression of arginase and production of NO by immature myeloid cells. The in vivo clearance of a bacterial infection was not impaired. DCs demonstrated reduced IL‐12 production and altered antigen presentation, resulting in decreased proliferation but enhanced IFN‐γ production by CD4+ cells. CD4+ T cells from mice immunized on day 2 after CLP showed reduced Th1 and Th2 cytokine production. In addition, there was an increase in Treg cells. Interestingly, levels of immature B cells decreased but levels of mature B cells increased two days after CLP. However, adoptive transfer of naïve CD4+ T cells, naïve B cells, or naïve DCs did not rescue the antigen‐specific antibody response.

Chronic psychosocial stress increases the risk for inflammation-related colon carcinogenesis in male mice

Patients with inflammatory bowel diseases (IBDs) have a higher risk of developing colorectal cancer (CRC) than the general population. Furthermore, chronic psychosocial stress increases the likelihood of developing IBD and multiple types of malignant neoplasms, including CRC. Here, for the first time, we investigate the effects of chronic psychosocial stress in male mice on an artificially induced CRC, by employing the chronic subordinate colony (CSC) housing paradigm in combination with the reliable azoxymethane (AOM)/dextran sodium sulfate (DSS) CRC model. Colonoscopy revealed that CSC mice showed accelerated macroscopic suspect lesions. In addition, more CSC mice developed low-grade dysplasia (LGD) and/or high-grade dysplasia (HGD) in the colonic tissue compared to the single-housed control mice (SHC). CSC mice showed an increased number of Ki67+ and a decreased number of terminal deoxynucleotidyl transferase dUTP nick end labeling epithelial cells in colonic tissue. Colonic liver receptor homolog-1 (LRH-1), cyclooxygenase II (COXII), tumor necrosis factor, forkhead box P3 (FoxP3) mRNA as well as colonic ß-catenin, COXII, and LRH-1 protein expression were also increased in CSC compared with SHC mice. Although the number of CD4+ Th cells was increased, a tendency toward a decreased colonic interferon-γ (IFN-γ) mRNA expression was observed. Furthermore, despite an increased percentage of CD3+ cells and CD3+/FoxP3+ double-positive cells within mesenteric lymph node cells of CSC mice, IFN-γ secretion from these cells was unaffected. Altogether, our results suggest that chronic psychosocial stress increases the risk for AOM/DSS-induced and, thus, inflammation-related CRC. Finally, assessment of additional time points may test whether the shift from tumor-protective Th1 cell to regulatory T-cell immunity represents a consequence of increased carcinogenesis or a causal factor involved in its development.

Tracing Functional Antigen-Specific CCR6+ Th17 Cells after Vaccination

Background The function of T helper cell subsets in vivo depends on their location, and one hallmark of T cell differentiation is the sequential regulation of migration-inducing chemokine receptor expression. CC-chemokine receptor 6 (CCR6) is a trait of tissue-homing effector T cells and has recently been described as a receptor on T helper type 17 (Th17) cells. Th17 cells are associated with autoimmunity and the defence against certain infections. Although, the polarization of Th cells into Th17 cells has been studied extensively in vitro, the development of those cells during the physiological immune response is still elusive. Methodology/Principal Findings We analysed the development and functionality of Th17 cells in immune-competent mice during an ongoing immune response. In naïve and vaccinated animals CCR6+ Th cells produce IL-17. The robust homeostatic proliferation and the presence of activation markers on CCR6+ Th cells indicate their activated status. Vaccination induces antigen-specific CCR6+ Th17 cells that respond to in vitro re-stimulation with cytokine production and proliferation. Furthermore, depletion of CCR6+ Th cells from donor leukocytes prevents recipients from severe disease in experimental autoimmune encephalomyelitis, a model for multiple sclerosis in mice. Conclusions/Significance In conclusion, we defined CCR6 as a specific marker for functional antigen-specific Th17 cells during the immune response. Since IL-17 production reaches the highest levels during the immediate early phase of the immune response and the activation of Th17 cells precedes the Th1 cell differentiation we tent to speculate that this particular Th cell subset may represent a first line effector Th cell subpopulation. Interference with the activation of this Th cell subtype provides an interesting strategy to prevent autoimmunity as well as to establish protective immunity against infections.

TNF controls the infiltration of dendritic cells into the site of Leishmania major infection.

TNF-negative C57BL/6 (B6.TNF−/−) mice are highly susceptible to Leishmania (L.) major infection and succumb rapidly to fatal leishmaniasis. A T helper type 1 (Th1) cell-mediated immune response is central for protective anti-leishmanial immunity. Therefore, the observed susceptibility of B6.TNF−/− mice to L. major parasites could be caused by a deficiency in mounting a Th1 response. Analysis of infected footpads revealed, that B6.TNF−/− mice exhibited a substantially diminished formation of DCs at the site of infection. Furthermore, Th1 cytokines such as IFN-γ were reduced in footpads of infected B6.TNF−/− mice. Cutaneous reconstitution of B6.TNF−/− mice with either bone marrow derived DCs (BM-DCs) or recombinant TNF simultaneous to infection resulted in an increased expression of cytokines such as IFN-γ and in an enhanced presence of Leishmania-antigen in skin draining lymph nodes. In addition, the individual time of survival was doubled. In conclusion we demonstrate that the expression of dermal TNF is necessary to provide an environment that initiates a local inflammatory response, but is not sufficient to induce protective immunity.

TNF-dependent overexpression of CCL21 is an underlying cause of progressive lymphoaccumulation in generalized lymphoproliferative disorder

The human condition autoimmune lymphoproliferative syndrome and the murine mutation generalized lymphoproliferative disorder (gld/gld) are both caused by mutations of Fas or Fas ligand and are characterized by severe splenomegaly and lymphadenopathy. In the mouse, the additional absence of TNF attenuates the gld/gld syndrome through an unknown mechanism. We hypothesized that this unexpected outcome was not mediated by increased apoptosis but changes of T cell localization. We demonstrated that the homeostatic chemokine CCL21 is strongly up‐regulated in the spleen of C57BL/6 (B6).gld/gld and B6.gld/gld.TRAIL–/– mice. In contrast, a distinct consequence of TNF deficiency in B6.gld/gld mice was the substantially reduced splenic production of CCL21. An analysis of the cognate chemokine receptor CCR7 showed a complete, age‐dependent down‐regulation of this receptor on B6.gld/gld conventional peripheral T cells that are therefore unable to react to this chemokine. These results demonstrate a new role for the pro‐inflammatory cytokine TNF and the TNF‐regulated chemokine CCL21 in the complex etiology of the autoimmune syndrome in B6.gld/gld mice.

An early reduction in Treg cells correlates with enhanced local inflammation in cutaneous leishmaniasis in CCR6-deficient mice.

Resistance to Leishmania major infection is dependent on the development of a cell-mediated Th1 immune response in resistant C57BL/6 mice whereas Th2-prone BALB/c mice develop non-healing lesions after infection. The chemokine receptor CCR6 is shared by anti-inflammatory regulatory T cells and pro-inflammatory Th17 cells. In a recent study we showed that C57BL/6 mice deficient in CCR6 exhibited enhanced footpad swelling and impaired T helper cell migration indicated by reduced recruitment of total T helper cells into the skin after infection and a reduced delayed type hypersensitivity reaction. Based on these findings we tested whether the lack of CCR6 alters Treg or Th17 cell responses during the course of Leishmania major infection. When we analyzed T cell subsets in the lymph nodes of CCR6-deficient mice, Th17 cell numbers were not different. However, reduced numbers of Treg cells paralleled with a stronger IFNγ response. Furthermore, the early increase in IFNγ-producing cells correlated with increased local tissue inflammation at later time points. Our data indicate an important role of CCR6 for Treg cells and a redundant role for Th17 cells in a Th1 cell-driven anti-parasitic immune response against Leishmania major parasites in resistant C57BL/6 mice.

Age-dependent, polyclonal hyperactivation of T cells is reduced in TNF-negative gld/gld mice

The generalized lymphoproliferative disorder (gld) mouse strain is characterized by severe splenomegaly/lymphadenopathy, the production of autoimmune antibodies, and the appearance of CD4/CD8‐negative T cells. An additional TNF deficiency of gld/gld mice attenuates the course of the disorder through a yet‐unknown mechanism. In this study, we could demonstrate that the reduced splenomegaly and lymphadenopathy in B6.gld/gld.TNF−/− mice were correlated with a decreased peripheral T cell proliferation rate and a delayed polyclonal activation. A comparative analysis of naïve T cells and memory/effector T cells showed an age‐dependent difference in the T cell activation pattern in the spleen of B6.gld/gld and B6.gld/gld.TNF−/− mice. T cells from B6.gld/gld.TNF−/− spleens and lymph nodes showed significantly higher levels of CCR7 and CD62 ligand on their surface compared with B6.gld/gld mice when mice of the same age were compared. Additionally, we found an increased titer of the Th1 cytokine IFN‐γ in the serum of B6.gld/gld mice, whereas the concentration of IFN‐γ was markedly reduced in the serum of B6.gld/gld.TNF−/− mice. These findings support the hypothesis that increased T cell activation and proliferation in the presence of TNF contribute to the exacerbation of the gld syndrome.

Induction of Suppressor Cells and Increased Tumor Growth following Chronic Psychosocial Stress in Male Mice

To study the impact of psychosocial stress on the immune system, male mice were subjected to chronic subordinate colony housing (CSC), a preclinically validated mouse model for chronic psychosocial stress. CSC substantially affected the cell composition of the bone marrow, blood, and spleen by inducing myelopoiesis and enhancing the frequency of regulatory T cells in the CD4 population. Expansion of the myeloid cell compartment was due to cells identified as immature inflammatory myeloid cells having the phenotype of myeloid-derived suppressor cells of either the granulocytic or the monocytic type. Catecholaminergic as well as TNF signaling were implicated in these CSC-induced cellular shifts. Although the frequency of regulatory cells was enhanced following CSC, the high capacity for inflammatory cytokine secretion of total splenocytes indicated an inflammatory immune status in CSC mice. Furthermore, CSC enhanced the suppressive activity of bone marrow-derived myeloid-derived suppressor cells towards proliferating T cells. In line with the occurrence of suppressor cell types such as regulatory T cells and myeloid-derived suppressor cells, transplanted syngeneic fibrosarcoma cells grew better in CSC mice than in controls, a process accompanied by pronounced angiogenesis and clustering of immature myeloid cells in the tumor tissue. In addition, tumor implantation after CSC reinforced the CSC-induced increase in myeloid-derived suppressor cells and regulatory T cell frequencies while the CSC-induced cellular changes eased off in mice without tumor. Together, our data suggest a role for suppressor cells such as regulatory T cells and myeloid-derived suppressor cells in the enhanced tumor growth after chronic psychosocial stress.