The intracellular Gram-negative bacterium Francisella tularensis causes the disease tularemia and is known for its ability to subvert host immune responses. Previous work from our laboratory identified the PI3K/Akt pathway and SHIP as critical modulators of host resistance to Francisella. Here, we show that SHIP expression is strongly down-regulated in monocytes and macrophages following infection with F. tularensis novicida (F.n.). To account for this negative regulation we explored the possibility that microRNAs (miRs) that target SHIP may be induced during infection. There is one miR that is predicted to target SHIP, miR-155. We tested for induction and found that F.n. induced miR-155 both in primary monocytes/macrophages and in vivo. Using luciferase reporter assays we confirmed that miR-155 led to down-regulation of SHIP, showing that it specifically targets the SHIP 3′UTR. Further experiments showed that miR-155 and BIC, the gene that encodes miR-155, were induced as early as four hours post-infection in primary human monocytes. This expression was dependent on TLR2/MyD88 and did not require inflammasome activation. Importantly, miR-155 positively regulated pro-inflammatory cytokine release in human monocytes infected with Francisella. In sharp contrast, we found that the highly virulent type A SCHU S4 strain of Francisella tularensis (F.t.) led to a significantly lower miR-155 response than the less virulent F.n. Hence, F.n. induces miR-155 expression and leads to down-regulation of SHIP, resulting in enhanced pro-inflammatory responses. However, impaired miR-155 induction by SCHU S4 may help explain the lack of both SHIP down-regulation and pro-inflammatory response and may account for the virulence of Type A Francisella.

During infection with viruses that establish latency, the immune system needs to maintain lifelong control of the infectious agent in the presence of persistent Ag. By using a γ-herpesvirus (γHV) infection model, we demonstrate that a small number of virus-specific central-memory CD8+ T cells develop early during infection, and that virus-specific CD8+ T cells maintain functional and protective capacities during chronic infection despite low-level Ag persistence. During the primary immune response, we show generation of CD8+ memory T cell precursors expressing lymphoid homing molecules (CCR7, L-selectin) and homeostatic cytokine receptors (IL-7α, IL-2/IL-15β). During long-term persistent infection, central-memory cells constitute 20–50% of the virus-specific CD8+ T cell population and maintain the expression of L-selectin, CCR7, and IL-7R molecules. Functional analyses demonstrate that during viral persistence: 1) CD8+ T cells maintain TCR affinity for peptide/MHC complexes, 2) the functional avidity of CD8+ T cells measured as the capacity to produce IFN-γ is preserved intact, and 3) virus-specific CD8+ T cells have in vivo killing capacity. Next, we demonstrate that at 8 mo post-virus inoculation, long-term CD8+ T cells are capable of mediating a protective recall response against the establishment of γHV68 splenic latency. These observations provide evidence that functional CD8+ memory T cells can be generated and maintained during low-load γHV68 persistence.

Background: Human monocyte inflammatory responses differ between virulent and attenuated Francisella infection.Results: A mixed infection model showed that the virulent F. tularensis Schu S4 can attenuate inflammatory cytokine responses to the less virulent F. novicida in human monocytes.Conclusion: F. tularensis dampens inflammatory response by an active process.Significance: This suppression may contribute to enhanced pathogenicity of F. tularensis. Francisella tularensis is a Gram-negative facultative bacterium that can cause the disease tularemia, even upon exposure to low numbers of bacteria. One critical characteristic of Francisella is its ability to dampen or subvert the host immune response. Previous work has shown that monocytes infected with highly virulent F. tularensis subsp. tularensis strain Schu S4 responded with a general pattern of quantitatively reduced pro-inflammatory signaling pathway genes and cytokine production in comparison to those infected with the less virulent related F. novicida. However, it has been unclear whether the virulent Schu S4 was merely evading or actively suppressing monocyte responses. By using mixed infection assays with F. tularensis and F. novicida, we show that F. tularensis actively suppresses monocyte pro-inflammatory responses. Additional experiments show that this suppression occurs in a dose-dependent manner and is dependent upon the viability of F. tularensis. Importantly, F. tularensis was able to suppress pro-inflammatory responses to earlier infections with F. novicida. These results lend support that F. tularensis actively dampens human monocyte responses and this likely contributes to its enhanced pathogenicity.

Increased treatment complexity in patients with diabetes contributes to medication nonadherence. Patient portals that are accessible through electronic health records may offer improved patient-provider communication and better medication management in patients with diabetes and uncontrolled hypertension. We conducted a prospective, mixedmethods evaluation of the Duke PillBox application, a SMART-on-FHIR medication management application integrated into the electronic health record patient portal. Adults with active portal status, diabetes, and uncontrolled hypertension participated in a usability/feasibility survey and communicated with a pharmacist via the portal-based application who conducted medication reconciliation and discussed medication indications, side effects, and barriers to use with patients. Of the eligible patients (n = 285), 29 (10%) were interested, and 12 (8%) participated. Challenges to usability were due to the electronic health record-portal communication interface (91%), browser access and compatibility (55%), and persistent lists of unused medications in the electronic health record (27%). The findings of this study suggest that electronic health record-integrated medication applications are desirable as indicated in patient interactions at the outset and conclusion of the study. Persistent challenges included electronic health record integration, interoperability, user interface, and browser connectivity for both patients and providers.