Toll-Like Receptor 3 Signaling on Macrophages Is Required for Survival Following Coxsackievirus B4 Infection

Toll-like receptor 3 (TLR3) has been proposed to play a central role in the early recognition of viruses by sensing double stranded RNA, a common intermediate of viral replication. However, several reports have demonstrated that TLR3 signaling is either dispensable or even harmful following infection with certain viruses. Here, we asked whether TLR3 plays a role in the response to coxsackievirus B4 (CB4), a prevalent human pathogen that has been associated with pancreatitis, myocarditis and diabetes. We demonstrate that TLR3 signaling on macrophages is critical to establish protective immunity to CB4. TLR3 deficient mice produced reduced pro-inflammatory mediators and are unable to control viral replication at the early stages of infection resulting in severe cardiac damage. Intriguingly, the absence of TLR3 did not affect the activation of several key innate and adaptive cellular effectors. This suggests that in the absence of TLR3 signaling on macrophages, viral replication outpaces the developing adaptive immune response. We further demonstrate that the MyD88-dependent signaling pathways are not only unable to compensate for the loss of TLR3, they are also dispensable in the response to this RNA virus. Our results demonstrate that TLR3 is not simply part of a redundant system of viral recognition, but rather TLR3 plays an essential role in recognizing the molecular signatures associated with specific viruses including CB4

Immunomodulation of Antigen Presenting Cells Promotes Natural Regulatory T Cells That Prevent Autoimmune Diabetes in NOD Mice

Progression towards type 1 diabetes (T1D) in susceptible patients is linked to a progressive decline in the capacity of regulatory T cells (Treg) to maintain tolerance. As such, therapies aimed at redressing the failing Treg compartment have been the subject of intense investigation. Treg dysfunction in T1D has recently been linked to a reduced capacity of antigen presenting cells (APCs) to maintain Treg function rather than Treg intrinsic defects. This suggests that therapies aimed simply at addressing the failing Treg compartment are unlikely to provide long-term protection. Here, we demonstrate that modulation of the inflammatory status of CD11b+CD11c2 APCs favors the upregulation of protective Tregs in a mouse model of T1D. We further demonstrate that reduced expression of the costimulatory molecule CD40 plays a role in this increased immunoregulatory capacity. Strikingly, Treg upregulation resulted exclusively from an increase in natural Tregs rather than the peripheral conversion of conventional T cells. This suggests that modulation of CD11b+ CD11c2 APCs inflammatory properties favors the establishment of natural Treg responses that, unlike adaptive Treg responses, are likely to maintain tolerance to a broad range of antigens. As such, modulation of this APC subset represents a potentia

CB4 infection in the context of TGF-β does not induce conversion of naïve T cells to adaptive Tregs but rather increases the levels of natural Tregs.

<p>A) Representative flow cytometry plots of Foxp3 and Helios expression by CD4+ T cells from the PLNs of mock-infected (left panel) or CB4-infected NODTGFβ mice at day 7PI. B) Average percentage of Helios+ cells among CD4+Foxp3+ Tregs from the PLNs of NODTGFβ mice with increased Treg levels at day 7 PI with CB4 (black bars) or mock-infection with DMEM (white bars). C) Average percentage of Helios- cells among CD4+Foxp3+ Tregs from the PLNs of NODTGFβ mice with increased Treg levels at day 7 PI with CB4 (black bars) or mock-infection with DMEM (white bars). Data represent mean + s.e.m from at least 2 separate experiments (n = at least 4 per group). D) Representative flow cytometry plots of CD4 and GFP (Foxp3) expression of donor (Thy1.1+) cells recovered from the spleen (left panels) or PLNs (right panels) of NOD (top panels) or NODTGF-β (bottom panels) recipient mice at day 7 following infection with CB4. Data is representative of 2 separate experiments, n = 3 for NOD recipients and n = 6 for NODTGFβ recipients.</p

Absence of CD40 on CD11b+CD11c− APCs is sufficient to increase Treg levels following CB4 infection.

<p>Average percentage of CD4 T cells expressing Foxp3 in the A) PLNs or B) pancreas of NODCD40KO mice mock-infected with DMEM (white bars) or infected with CB4 (black bars) (n = at least 10 per group). C) Average percentage of CD4+ T cells expressing Foxp3 in the PLNs of uninfected 10–12 week old NOD mice mock-transferred with DMEM (white bar) or adoptively transferred with CD11b+CD11c− FACS sorted from CB4 infected NODCD40KO (black bar) or mock-infected NODCD40KO mice (grey bars). Data represent mean + s.e.m from at least 3 separate experiments (n = at least 7 per group).</p

CD11b+CD11c− APCs produce lower levels of inflammatory cytokines following CB4 infection in the context of TGF-β.

<p><i>Ex vivo</i> production of A, B) TNF-α or C,D) IL-6 from CD11b+CD11c− APCs FACS sorted from CB4 infected NOD (black bar) or NODTGFβ (white bar) mice. Cytokine levels were measured from culture supernatants following 24 hours of incubation. Panels A,C are representative data from one experiment with pooled mice of each genotype while panels B,D represent mean + s.e.m of cytokine levels from 6 separate experiments normalized to the cytokine levels produced by WT NOD mice infected with CB4 in each separate experiments.</p