Glennys V. Reynoso’s research while affiliated with National Institute of Allergy and Infectious Diseases, National Institutes of Health and other places

Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell-T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we find that during the first 24 h of infection, CHIKV RNA accumulates in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response, including recruitment of myeloid cells to the LN, was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, we find that antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.

There are no directed therapies to the latency program of the human gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus. Activated host factor signal transducer and activator of transcription 3 (STAT3) is a hallmark of cancers caused by these viruses. We applied the murine gammaherpesvirus pathogen system to explore STAT3 function upon primary B cell infection in the host. Since STAT3 deletion in all CD19+ B cells of infected mice led to altered B and T cell responses, we generated chimeric mice with both normal and STAT3-deleted B cells. B cells lacking STAT3 failed to support virus latency compared to normal B cells from the same infected animal. Loss of STAT3 impaired B cell proliferation and differentiation and led to a striking upregulation of interferon-stimulated genes. These findings expand our understanding of STAT3-dependent processes that are key to its function as a pro-viral latency determinant for oncogenic gammaherpesviruses in B cells and may provide novel therapeutic targets.

Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell-T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we find that during the first 24 h of infection, CHIKV RNA accumulates in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response, including recruitment of myeloid cells to the LN, was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, we find that antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.

Innate lymphoid cells (ILCs) are a diverse group of tissue-resident lymphocytes that lack antigen-specific receptors commonly found on adaptive immune cells. While many different types of ILCs have been described, only group 1 ILCs (ILC1s) are known to exhibit marked antiviral activity. We recently described ILC1-mediated protection of the oral mucosa, a critical barrier tissue and site of continued pathogen exposure. Based on these studies, we hypothesized that these innate antiviral cells might also respond to inflammation induced by a distal viral infection. To address this, we used a common mouse model of vaccinia virus (VACV) infection of the peritoneal cavity, which spreads locally but does not result in infection of the oral mucosa. Confocal microscopy of frozen sections of the lip demonstrated a marked increase in the number and density of oral mucosal ILC1s in response to distal infection. Further, ILC1s remained sustainably elevated for weeks following the initial infection. Ongoing experiments will assess the ability of this enhanced layer of ILC1s to provide heightened antiviral immunity in the oral mucosa and determine the timeframes in which this occurs. Together, our data suggest that the ability of ILC1s to globally sense infection and enhance protection in response to distal infection may represent a previously unappreciated mechanism of trained immunity in the tissue.

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor STAT3. To better understand the role of STAT3 during gammaherpesvirus latency and immune control, we utilized murine gammaherpesvirus 68 (MHV68) infection. Genetic deletion of STAT3 in B cells of CD19 cre/+ Stat3 f/f mice reduced peak latency approximately 7-fold. However, infected CD19 cre/+ Stat3 f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to WT littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeras consisting of WT and STAT3-knockout B cells. Using a competitive model of infection, we discovered a dramatic reduction in latency in STAT3-knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that STAT3 promotes proliferation and B cell processes of the germinal center but does not directly regulate viral gene expression. Last, this analysis uncovered a STAT3-dependent role for dampening type I IFN responses in newly infected B cells. Together, our data provide mechanistic insight into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses. IMPORTANCE There are no directed therapies to the latency program of the gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus. Activated host factor STAT3 is a hallmark of cancers caused by these viruses. We applied the murine gammaherpesvirus pathogen system to explore STAT3 function upon primary B cell infection in the host. Since STAT3 deletion in all CD19+ B cells of infected mice led to altered B and T cell responses, we generated chimeric mice with both normal and STAT3-deleted B cells. B cells lacking STAT3 failed to support virus latency compared to normal B cells from the same infected animal. Loss of STAT3 impaired B cell proliferation and differentiation and led to a striking upregulation of interferon-stimulated genes. These findings expand our understanding of STAT3-dependent processes key to its function as a pro-viral latency determinant for oncogenic gammaherpesviruses in B cells and may provide novel therapeutic targets.

To disseminate through the body, Zika virus (ZIKV) is thought to exploit the mobility of myeloid cells, in particular monocytes and dendritic cells. However, the timing and mechanisms underlying shuttling of the virus by immune cells remains unclear. To understand the early steps in ZIKV transit from the skin, at different time points, we spatially mapped ZIKV infection in lymph nodes (LNs), an intermediary site en route to the blood. Contrary to prevailing hypotheses, migratory immune cells are not required for the virus to reach the LNs or blood. Instead, ZIKV rapidly infects a subset of sessile CD169+ macrophages in the LNs, which release the virus to infect downstream LNs. Infection of CD169+ macrophages alone is sufficient to initiate viremia. Overall, our experiments indicate that macrophages that reside in the LNs contribute to initial ZIKV spread. These studies enhance our understanding of ZIKV dissemination and identify another anatomical site for potential antiviral intervention.

The success of poxviruses as pathogens depends upon their antagonism of host responses by multiple immunomodulatory proteins. The largest of these expressed by ectromelia virus (the agent of mousepox) is C15, one member of a well-conserved poxviral family previously shown to inhibit T cell activation. Here, we demonstrate by quantitative immunofluorescence imaging that C15 also limits contact between natural killer (NK) cells and infected cells in vivo. This corresponds to an inhibition in the number of total and degranulating NK cells, ex vivo and in vitro, with no detectable impact on NK cell cytokine production nor the transcription of factors related to NK cell recruitment or activation. Thus, in addition to its previously identified capacity to antagonize CD4 T cell activation, C15 inhibits NK cell cytolytic function, which results in increased viral replication and dissemination in vivo. This work builds on a body of literature demonstrating the importance of early restriction of virus within the draining lymph node.

Disseminated coccidioidomycosis (DCM) is caused by Coccidioides, pathogenic fungi endemic to the Southwestern United States and Mexico. Illness occurs in approximately 30% of those infected, <1% of whom develop disseminated disease. To address why some individuals allow dissemination, we enrolled DCM patients and performed whole-exome sequencing. In an exploratory set of 67 DCM patients, two had haploinsufficient STAT3 mutations, while defects in β-glucan sensing and response were seen in 34/67 (50.7%) cases. Damaging CLEC7A (n=14) and PLCG2 (n=11) variants were associated with impaired production of β-glucan-stimulated TNF-α from peripheral blood mononuclear cells compared to healthy controls (P<0.005). Using ancestry-matched controls, damaging CLEC7A and PLCG2 variants were over-represented in DCM (P=0.0206, P=0.015, respectively) including CLEC7A Y238* (P=0.0105) and PLCG2 R268W (P=0.0025). A validation cohort of 111 DCM patients confirmed PLCG2 R268W (P=0.0276), CLEC7A I223S (P=0.044), and CLEC7A Y238* (P=0.0656). Stimulation with a DECTIN-1 agonist induced DUOX1/DUOXA1-derived H2O2 in transfected cells. Heterozygous DUOX1 or DUOXA1 variants which impaired H2O2 production were overrepresented in discovery and validation cohorts. Patients with DCM have impaired β-glucan sensing or response affecting TNF-α and H2O2 production. Impaired Coccidioides recognition and decreased cellular response are associated with disseminated coccidioidomycosis.

Zika virus (ZIKV) is an arthopod-vectored flavivirus that disseminates from the infection site into peripheral tissues, where it can elicit virus-induced pathology. To move through the body, ZIKV is thought to exploit the mobility of myeloid cells, in particular monocytes and dendritic cells. However, multiple distinct steps during viral spread culminate in peripheral tissue infection, and the timing and mechanisms underlying mobile immune cell shuttling of virus remain unclear. To understand the very early steps in ZIKV dissemination from the skin, we kinetically and spatially mapped ZIKV-infected lymph nodes (LNs), an intermediary stop en route to the blood. Contrary to dogma, migratory immune cells were not required for large quantities of virus to reach the LN or blood. Instead, ZIKV rapidly infected a subset of immobile macrophages in the LN, which shed virus through the lymphatic pathway into the blood. Importantly, infection of LN macrophages alone was sufficient to initiate viremia. Together, our studies indicate that sessile macrophages that live and die in the LN contribute to initial ZIKV spread to the blood. These data build a more complete picture of ZIKV movement through the body and identify an alternate anatomical site for potential antiviral intervention. Highlights ZIKV infects and replicates in distinct LN macrophage populations LN macrophage infection results in infectious virus in the blood Virus reaches the blood in the absence of DC migration or monocyte infection Nodal macrophage infection does not sustain viremia or induce morbidity Graphical abstract Caption ZIKV infects lymph node macrophages, which shed infectious virus into the lymph and then blood.

The success of poxviruses as pathogens depends upon their antagonism of host responses by multiple immunomodulatory proteins. The largest of these expressed by ectromelia virus (the agent of mousepox) is C15, one member of a well-conserved poxviral family previously shown to inhibit T cell activation. Here, we demonstrate by quantitative immunofluorescence imaging that C15 also limits contact between natural killer (NK) cells and infected cells in vivo . This corresponds to an inhibition in the number of total and degranulating NK cells, ex vivo and in vitro , with no detectable impact on NK cell cytokine production nor the transcription of factors related to NK cell recruitment or activation. Thus, in addition to its previously identified capacity to antagonize CD4 T cell activation, C15 inhibits NK cell cytolytic function, which results in increased viral replication and dissemination in vivo . This work builds on a body of literature demonstrating the importance of early restriction of virus within the draining lymph node. Summary Poxvirus B22 family proteins are important virulence factors known to inhibit T cell functions. Peauroi et al. identify a novel function of the ectromelia virus homolog, C15, which inhibits NK cell-target contact and cytolytic function to facilitate early viral spread. (Provide a short, ∼40-word summary statement for the online JEM table of contents and alerts. This summary should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person.)