Autocrine IL-10 Signaling Promotes Dendritic Cell Type-2 Activation and Persistence of Murine Cryptococcal Lung Infection.

The substantial morbidity and mortality caused by invasive fungal pathogens, including Cryptococcus neoformans, necessitates increased understanding of protective immune responses against these infections. Our previous work using murine models of cryptococcal lung infection demonstrated that dendritic cells (DCs) orchestrate critical transitions from innate to adaptive immunity and that IL-10 signaling blockade improves fungal clearance. To further understand interrelationships among IL-10 production, fungal clearance, and the effect of IL-10 on lung DCs, we performed a comparative temporal analysis of cryptococcal lung infection in wild type C57BL/6J mice (designated IL-10+/+) and IL-10-/- mice inoculated intratracheally with C. neoformans (strain 52D). Early and sustained IL-10 production by lung leukocytes was associated with persistent infection in IL-10+/+ mice, whereas fungal clearance was improved in IL-10-/- mice during the late adaptive phase of infection. Numbers of monocyte-derived DCs, T cells, and alveolar and exudate macrophages were increased in lungs of IL-10-/- versus IL-10+/+ mice concurrent with evidence of enhanced DC type-1, Th1/Th17 CD4 cell, and classical macrophage activation. Bone marrow-derived DCs stimulated with cryptococcal mannoproteins, a component of the fungal capsule, upregulated expression of IL-10 and IL-10R, which promoted DC type-2 activation in an autocrine manner. Thus, our findings implicate fungus-triggered autocrine IL-10 signaling and DC type-2 activation as important contributors to the development of nonprotective immune effector responses, which characterize persistent cryptococcal lung infection. Collectively, this study informs and strengthens the rationale for IL-10 signaling blockade as a novel treatment for fungal infections.

Anti-PD-1 Antibody Treatment Promotes Clearance of Persistent Cryptococcal Lung Infection in Mice.

Activation of immunomodulatory pathways in response to invasive fungi can impair clearance and promote persistent infections. The programmed cell death protein-1 (PD-1) signaling pathway inhibits immune effector responses against tumors, and immune checkpoint inhibitors that block this pathway are being increasingly used as cancer therapy. The objective of this study was to investigate whether this pathway contributes to persistent fungal infection and to determine whether anti-PD-1 Ab treatment improves fungal clearance. Studies were performed using C57BL/6 mice infected with a moderately virulent strain of Cryptococcus neoformans (52D), which resulted in prolonged elevations in fungal burden and histopathologic evidence of chronic lung inflammation. Persistent infection was associated with increased and sustained expression of PD-1 on lung lymphocytes, including a mixed population of CD4+ T cells. In parallel, expression of the PD-1 ligands, PD-1 ligands 1 and 2, was similarly upregulated on specific subsets of resident and recruited lung dendritic cells and macrophages. Treatment of persistently infected mice for 4 wk by repetitive administration of neutralizing anti-PD-1 Ab significantly improved pulmonary fungal clearance. Treatment was well tolerated without evidence of morbidity. Immunophenotyping revealed that anti-PD-1 Ab treatment did not alter immune effector cell numbers or myeloid cell activation. Treatment did reduce gene expression of IL-5 and IL-10 by lung leukocytes and promoted sustained upregulation of OX40 by Th1 and Th17 cells. Collectively, this study demonstrates that PD-1 signaling promotes persistent cryptococcal lung infection and identifies this pathway as a potential target for novel immune-based treatments of chronic fungal disease.

Bovine Leukemia Virus and Mycobacterium avium subsp. paratuberculosis Are Not Associated with Shiga Toxin-Producing Escherichia coli Shedding in Cattle.

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis in cattle, and Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in cattle. Both diseases are chronic in nature and can lead to the disruption of normal immunological or physiological processes. Cattle are the major reservoir of Shiga toxin-producing Escherichia coli (STEC), a cause of foodborne illness in humans. We tested the hypothesis that cattle infected with BLV or MAP are more likely to shed STEC. We conducted a cross-sectional study during the summers of 2011 and 2012 in 11 Michigan cattle herds. A fecal sample from each animal was collected for STEC culture, and multiplex PCR for stx1, stx2, and eaeA was used to screen suspect colonies for STEC confirmation. Antibody detection enzyme-linked immunosorbent assays for BLV and MAP were used to screen serum from each animal. Flow cytometry was used to quantify the percentage of lymphocytes, monocytes, and neutrophils in a subsample (n =497) of blood samples. Of the animals sampled, 34.9% were BLV positive, 2.7% were MAP positive, and 16% were shedding STEC. Cattle in the dairy herds had a higher frequency of BLV and MAP than did those in beef herds, but more cattle in beef herds were shedding STEC. Neither BLV nor MAP was associated with STEC shedding (P values of 0.6838 and 0.3341, respectively). We also observed no association between STEC status and the percentage of neutrophils (P value of 0.3565), lymphocytes (P value of 0.8422), or the lymphocyte-to-monocyte ratio (P value of 0.1800). Although controlling both BLV and MAP is important for overall herd health and productivity, we found no evidence that controlling BLV and MAP has an impact on STEC shedding in cattle.

Immunoregulation in Fungal Diseases.

This review addresses specific regulatory mechanisms involved in the host immune response to fungal organisms. We focus on key cells and regulatory pathways involved in these responses, including a brief overview of their broader function preceding a discussion of their specific relevance to fungal disease. Important cell types discussed include dendritic cells and regulatory T cells, with a focus on specific studies relating to their effects on immune responses to fungi. We highlight the interleukin-10, programmed cell death 1, and cytotoxic T lymphocyte-associated protein 4 signaling pathways and emphasize interrelationships between these pathways and the regulatory functions of dendritic cells and regulatory T cells. Throughout our discussion, we identify selected studies best illustrating the role of these cells and pathways in response to specific fungal pathogens to provide a contextual understanding of the tightly-controlled network of regulatory mechanisms critical to determining the outcome of exposure to fungal pathogens. Lastly, we discuss two unique phenomena relating to immunoregulation, protective tolerance and immune reactivation inflammatory syndrome. These two clinically-relevant conditions provide perspective as to the range of immunoregulatory mechanisms active in response to fungi.

Regulatory T cells and immune profiling in johne's disease lesions.

Johne's disease, caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic wasting disease of ruminants. Hallmark symptoms of clinical Johne's disease include diarrhea, progressive weight loss, and premature death; symptoms due largely to chronic inflammation in the small intestine. MAP colonizes resident macrophages within the ileum of the small intestine, subsequently establishing a persistent infection in the host. It has been proposed that regulatory T cells may play a role in the progression of Johne's disease, either through promotion of tolerance to MAP or via a loss in homeostasis that subsequently allows widespread inflammation. In this report, we evaluated the presence of Tregs, as well as other immune parameters, in the ileum and draining lymph nodes of MAP associated lesions. A lesion classification scheme was developed to categorize severity of MAP-induced lesions within infected tissues and subsequently regulatory T cell presence and overall immune activity were assessed corresponding to lesions of varying severity, in comparison to tissues from healthy control animals. Our results revealed a relationship between animal health and overall lesion severity within the infected tissues, as well as a relationship between bacterial burden and severity of pathology. Regulatory T cell abundance was shown to decrease with increasing lesion severity. Within the ileum, the expression of many Th1, Th2, and Treg-associated genes increased in mild lesions and decreased in severe lesions, whereas in the lymph nodes the expression of these genes tended to increase with increasing lesion severity. Based on our results, we conclude that a local loss of T cell (including Treg) activity occurs within severe ileal lesions associated with MAP, resulting in a loss of homeostasis that ultimately leads to the progression of clinical Johne's disease.

Regulatory T Cell Activity and Signs of T Cell Unresponsiveness in Bovine Paratuberculosis.

Johne's disease, caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP), is a wasting disease of ruminants displaying a long subclinical stage of infection followed by clinical disease characterized by severe diarrhea, wasting, and premature death. Immunologically, subclinical disease is characterized by a Th1 response effective at controlling intracellular infections such as that caused by MAP. In late subclinical disease, the Th1 response subsides and a non-protective Th2 response becomes prominent. One hypothesis for this shift in immune paradigm is that a population of MAP-reactive regulatory T cells (Tregs) develops during subclinical infection, limiting Th1-type responses to MAP antigens. To investigate this, we sought to accomplish the following: (1) determine if CD4(+)CD25(-) T cells exposed to MAP-infected macrophages develop a Treg phenotype, (2) develop a method to expand the relative abundance of Tregs in bovine peripheral blood lymphocyte populations, and (3) identify functional activities of expanded Tregs when combined with autologous peripheral blood mononuclear cells (PBMCs) and live MAP. We found that CD4(+)CD25(-) T cells exposed to MAP-infected macrophages from cows with Johne's disease do not show signs of a Treg phenotype and appear unresponsive to MAP antigens. A method for Treg expansion was successfully developed; however, based on results obtained in the subsequent functional studies it appears that these Tregs are not MAP-specific. Overall, it seems that T cell unresponsiveness, rather than Treg activity, is driving the Th1-to-Th2 immune shift observed during Johne's disease. Further, we have successfully developed a method to enrich non-specific bovine Tregs that exert suppressive effects against Th1 cytokine production.