Crosstalk between CD64+MHCII+ macrophages and CD4+ T cells drives joint pathology during chikungunya.

Communications between immune cells are essential to ensure appropriate coordination of their activities. Here, we observed the infiltration of activated macrophages into the joint-footpads of chikungunya virus (CHIKV)-infected animals. Large numbers of CD64+MHCII+ and CD64+MHCII- macrophages were present in the joint-footpad, preceded by the recruitment of their CD11b+Ly6C+ inflammatory monocyte precursors. Recruitment and differentiation of these myeloid subsets were dependent on CD4+ T cells and GM-CSF. Transcriptomic and gene ontology analyses of CD64+MHCII+ and CD64+MHCII- macrophages revealed 89 differentially expressed genes, including genes involved in T cell proliferation and differentiation pathways. Depletion of phagocytes, including CD64+MHCII+ macrophages, from CHIKV-infected mice reduced disease pathology, demonstrating that these cells play a pro-inflammatory role in CHIKV infection. Together, these results highlight the synergistic dynamics of immune cell crosstalk in driving CHIKV immunopathogenesis. This study provides new insights in the disease mechanism and offers opportunities for development of novel anti-CHIKV therapeutics.

Differential mucosal tropism and dissemination of classical and hypervirulent Klebsiella pneumoniae infection.

Klebsiella pneumoniae (Kp) infection is an important healthcare concern. The ST258 classical (c)Kp strain is dominant in hospital-acquired infections in North America and Europe, while ST23 hypervirulent (hv)Kp prevails in community-acquired infections in Asia. This study aimed to develop symptomatic mucosal infection models in mice that mirror natural infections in humans to gain a deeper understanding of Kp mucosal pathogenesis. We showed that cKp replicates in the nasal cavity instead of the lungs, and this early infection event is crucial for the establishment of chronic colonization in the cecum and colon. In contrast, hvKp replicates directly in the lungs to lethal bacterial load, and early infection of esophagus supported downstream transient colonization in the ileum and cecum. Here, we have developed an in vivo model that illuminates how differences in Kp tropism are responsible for virulence and disease phenotype in cKp and hvKp, providing the basis for further mechanistic study.

CD8+ T Cells Trigger Auricular Dermatitis and Blepharitis in Mice after Zika Virus Infection in the Absence of CD4+ T Cells.

Zika virus (ZIKV) became a public health concern when it re-emerged in 2015 owing to its ability to cause congenital deformities in the fetus and neurological complications in adults. Despite extensive data on protection, the interplay of protective and pathogenic adaptive immune responses toward ZIKV infection remains poorly understood. In this study, using a T-cell‒deficient mouse model that retains persistent ZIKV viral titers in the blood and organs, we show that the adoptive transfer of CD8+ T cells led to a significant reduction in viral load. This mouse model reveals that ZIKV can induce grossly visible auricular dermatitis and blepharitis, mediated by ZIKV-specific CD8+ T cells. Single-cell RNA sequencing of these causative CD8+ T cells from the ears shows an overactivated and elevated cytotoxic signature in mice with severe symptoms. Our results strongly suggest a role for CD8+ T-cell‒associated pathologies after ZIKV infection in CD4+ T-cell‒immunodeficient patients.

Malaria abrogates O'nyong-nyong virus pathologies by restricting virus infection in nonimmune cells.

O'nyongnyong virus (ONNV) is a re-emerging alphavirus previously known to be transmitted by main malaria vectors, thus suggesting the possibility of coinfections with arboviruses in co-endemic areas. However, the pathological outcomes of such infections remain unknown. Using murine coinfection models, we demonstrated that a preexisting blood-stage Plasmodium infection suppresses ONNV-induced pathologies. We further showed that suppression of viremia and virus dissemination are dependent on Plasmodium-induced IFNγ and are associated with reduced infection of CD45- cells at the site of virus inoculation. We further proved that treatment with IFNγ or plasma samples from Plasmodium vivax-infected patients containing IFNγ are able to restrict ONNV infection in human fibroblast, synoviocyte, skeletal muscle, and endothelial cell lines. Mechanistically, the role of IFNγ in restricting ONNV infection was confirmed in in vitro infection assays through the generation of an IFNγ receptor 1 α chain (IFNγR1)-deficient cell line.

Longitudinal [18F]FB-IL-2 PET Imaging to Assess the Immunopathogenicity of O'nyong-nyong Virus Infection.

O'nyong-nyong virus (ONNV) is an arthritogenic alphavirus that caused two large epidemics in 1959 and 1996, affecting millions of people in Africa. More recently, sero-surveillance of healthy blood donors conducted in 2019 revealed high rates of unreported ONNV infection in Uganda. Due to similar clinical symptoms with other endemic mosquito-borne pathogens in the region, including chikungunya virus, dengue virus and malaria, ONNV infections are often un- or misdiagnosed. Elucidating the immunopathogenic factors of this re-emerging arbovirus is critical with the expanding geographic distribution of competent vectors. This study reports the establishment of an immune competent C57BL6/J mouse model to mechanistically characterize ONNV infection and assess potential treatment efficacy. This mouse model successfully recapitulated arthralgia and viremia profiles seen in ONNV patients. Furthermore, longitudinal in-vivo PET imaging with [18F]FB-IL-2 (CD25+CD4+ binding probe) and histopathological assessment in this model demonstrated the pathogenic role of CD4+ T cells in driving joint pathology. Concordantly, in vivo CD4+ T cell depletion, or suppression with fingolimod, an FDA-approved immunomodulating drug, abrogated CD4+ T cell-mediated disease. This study demonstrates the importance of this immune competent ONNV model for future studies on factors influencing disease pathogenesis, which could shape the discovery of novel therapeutic strategies for arthritogenic alphaviruses.

Type I interferon shapes the quantity and quality of the anti-Zika virus antibody response.

OBJECTIVES: Zika virus (ZIKV) is a mosquito-borne flavivirus that re-emerged in 2015. The association between ZIKV and neurological complications initiated the development of relevant animal models to understand the mechanisms underlying ZIKV-induced pathologies. Transient inhibition of the type I interferon (IFN) pathway through the use of an IFNAR1-blocking antibody, MAR1-5A3, could efficiently permit active virus replication in immunocompetent animals. Type I IFN signalling is involved in the regulation of humoral responses, and thus, it is crucial to investigate the potential effects of type I IFN blockade towards B-cell responses. METHODS: In this study, comparative analysis was conducted using serum samples collected from ZIKV-infected wild-type (WT) animals either administered with or without MAR1-5A3. RESULTS: Serological assays revealed a more robust ZIKV-specific IgG response and subtype switching upon inhibition of type I IFN due to the abundance of antigen availability. This observation was corroborated by an increase in germinal centres, plasma cells and germinal centre B cells. Interestingly, although both groups of animals recognised different B-cell linear epitopes in the E and NS1 regions, there was no difference in neutralising capacity. Further characterisation of these epitopes in the E protein revealed a detrimental role of antibodies that were generated in the absence of type I IFN. CONCLUSION: This study highlights the role of type I IFN in shaping the anti-ZIKV antibody response to generate beneficial antibodies and will help guide development of better vaccine candidates triggering efficient neutralising antibodies and avoiding detrimental ones.

Shigella impairs human T lymphocyte responsiveness by hijacking actin cytoskeleton dynamics and T cell receptor vesicular trafficking.

Strategies employed by pathogenic enteric bacteria, such as Shigella, to subvert the host adaptive immunity are not well defined. Impairment of T lymphocyte chemotaxis by blockage of polarised edge formation has been reported upon Shigella infection. However, the functional impact of Shigella on T lymphocytes remains to be determined. Here, we show that Shigella modulates CD4+ T cell F-actin dynamics and increases cell cortical stiffness. The scanning ability of T lymphocytes when encountering antigen-presenting cells (APC) is subsequently impaired resulting in decreased cell-cell contacts (or conjugates) between the two cell types, as compared with non-infected T cells. In addition, the few conjugates established between the invaded T cells and APCs display no polarised delivery and accumulation of the T cell receptor to the contact zone characterising canonical immunological synapses. This is most likely due to the targeting of intracellular vesicular trafficking by the bacterial type III secretion system (T3SS) effectors IpaJ and VirA. The collective impact of these cellular reshapings by Shigella eventually results in T cell activation dampening. Altogether, these results highlight the combined action of T3SS effectors leading to T cell defects upon Shigella infection.

Pathogenic Th1 responses in CHIKV-induced inflammation and their modulation upon Plasmodium parasites co-infection.

The induction of polyarthritis and polyarthralgia is a hallmark of arthritogenic alphavirus infections, with an exceptionally higher morbidity observed with chikungunya virus (CHIKV). While the mechanisms underlying these incapacitating acute symptoms remain partially understood, the progression to chronic conditions in some cases remains unanswered. The highly pro-inflammatory nature of alphavirus disease has suggested the involvement of virus-specific, joint-infiltrating Th1 cells as one of the main pathogenic mediators of CHIKV-induced joint pathologies. This review summarizes the role of cell-mediated immune responses in CHIKV pathogenesis, with a specific focus on pro-inflammatory Th1 responses in the development of CHIKV joint inflammation. Furthermore, due to the explosive nature of arthritogenic alphavirus outbreaks and their recent expansion across the world, co-infections with other highly prevalent pathogens such as malaria are likely to occur but the pathological outcomes of such interactions in humans are unknown. This review will also discuss the potential impact of malaria co-infections on CHIKV pathogenesis and their relevance in alphavirus control programs in endemic areas.

Mutating chikungunya virus non-structural protein produces potent live-attenuated vaccine candidate.

Currently, there are no commercially available live-attenuated vaccines against chikungunya virus (CHIKV). Here, CHIKVs with mutations in non-structural proteins (nsPs) were investigated for their suitability as attenuated CHIKV vaccines. R532H mutation in nsP1 caused reduced infectivity in mouse tail fibroblasts but an enhanced type-I IFN response compared to WT-CHIKV Adult mice infected with this nsP-mutant exhibited a mild joint phenotype with low-level viremia that rapidly cleared. Mechanistically, ingenuity pathway analyses revealed a tilt in the anti-inflammatory IL-10 versus pro-inflammatory IL-1ß and IL-18 balance during CHIKV nsP-mutant infection that modified acute antiviral and cell signaling canonical pathways. Challenging CHIKV nsP-mutant-infected mice with WT-CHIKV or the closely related O'nyong-nyong virus resulted in no detectable viremia, observable joint inflammation, or damage. Challenged mice showed high antibody titers with efficient neutralizing capacity, indicative of immunological memory. Manipulating molecular processes that govern CHIKV replication could lead to plausible vaccine candidates against alphavirus infection.

Viperin controls chikungunya virus-specific pathogenic T cell IFNγ Th1 stimulation in mice.

Chikungunya virus (CHIKV) has been a worldwide threat since its reemergence in La Reunion Island in 2004. Expression of the interferon-stimulated protein Viperin correlates with viral load burden in patients, and studies in mice have demonstrated its role to limit disease severity against CHIKV infection. Using Viperin -/- mice, we aimed to understand the contribution of Viperin to the T-cell immune response against CHIKV. CD4 T-cell depletion in Viperin -/- mice showed that increased late acute joint inflammation (5-8 d postinfection) was exclusively mediated by T cells. Specifically, CHIKV-infected Viperin -/- mice showed an increased INFγ Th1 profile of CD4 T cells, enhanced INFγ stimulation by APCs, an increased INFγ secretion profile in the joint microenvironment, and increased numbers of inflammatory monocytes in virus-infected joints compared with WT mice. Bone marrow grafting experiments showed that Viperin expression in both hematopoietic and non-hematopoietic cells is instrumental in reducing disease severity associated with a CD4 T-cell response.

Multimodal assessments of Zika virus immune pathophysiological responses in marmosets.

Animal models that recapitulate the human pathophysiology have been developed as useful research tools. Although laboratory mice are widely used, they are phylogenetically "distant" to humans. New world monkeys, such as the common marmoset (Callithrix jacchus) have steadily gained prominence. In this report, marmosets are explored as an alternate in vivo model to investigate infection and immunity of Zika virus (ZIKV). Multimodal platforms, including ultrasound and magnetic resonance imaging (MRI), flow cytometry, and multiplex microbead immunoassays were established to comprehensively decipher immune responses and pathophysiological outcomes. While ZIKV-infected marmosets had detectable ZIKV RNA load in various body fluids, animals did not develop any observable lesions in their testes and brains as shown by ultrasound and MRI. Immune-phenotyping detected differences in the numbers of B cells, CD8+ T cells and HLADR+ NK cells during the first two weeks of infection. Neutralizing ZIKV-specific antibodies were elicited to high levels and targeted epitopes in the E protein. This study presents a one-stop-shop platform to study infection and pathophysiology in marmosets. While marmoset-specific research tools are being refined, the research values of these animals present them as a good model for immune-based therapies.

Plasmodium co-infection protects against chikungunya virus-induced pathologies.

Co-infection with Plasmodium and chikungunya virus (CHIKV) has been reported in humans, but the impact of co-infection on pathogenesis remains unclear. Here, we show that prior exposure to Plasmodium suppresses CHIKV-associated pathologies in mice. Mechanistically, Plasmodium infection induces IFNγ, which reduces viraemia of a subsequent CHIKV infection and suppresses tissue viral load and joint inflammation. Conversely, concomitant infection with both pathogens limits the peak of joint inflammation with no effect on CHIKV viraemia. Reduced peak joint inflammation is regulated by elevated apoptosis of CD4+ T-cells in the lymph nodes and disrupted CXCR3-mediated CD4+ T-cell migration that abolishes their infiltration into the joints. Virus clearance from tissues is delayed in both infection scenarios, and is associated with a disruption of B cell affinity-maturation in the spleen that reduces CHIKV-neutralizing antibody production.

Bacillus licheniformis in geogenic dust induces inflammation in respiratory epithelium.

Exposure to environmental geogenic (or earth-derived) dust can lead to more frequent and severe infections in the human airway. Particulate matter < 10 µm (PM10) is the component of air pollution that is commonly associated with the exacerbation of respiratory diseases. We have previously demonstrated that mice exposed to geogenic dust PM10 experienced an exacerbation of inflammatory responses to influenza A virus. Whether geogenic dust PM10 also exacerbates respiratory bacterial infection is not yet known, nor are the components of the dust that drive these responses. We treated airway bronchial epithelial cells (NuLi-1) with UV-irradiated geogenic dust PM10 from six remote Western Australian towns. High levels of IL-6 and IL-8 production were observed, as well as persistent microbial growth. 16 S rRNA sequencing of the growth identified the microbe as Bacillus licheniformis, a spore-forming, environmentally abundant bacterium. We next investigated the interaction of B. licheniformis with respiratory epithelium in vitro to determine whether this exacerbated infection with a bacterial respiratory pathogen (non-typeable Haemophilus influenzae, NTHi). Heat treatment (100 °C) of all PM10 samples eliminated B. licheniformis contamination and reduced epithelial inflammatory responses, suggesting that heat-labile and/or microbial factors were involved in the host response to geogenic dust PM10. We then exposed NuLi-1 epithelium to increasing doses of the isolated Bacillus licheniformis (multiplicity of infection of 10:1, 1:1 or 0.1:1 bacteria: cells) for 1, 3, and 24 h. B. licheniformis and NTHi infection (association and invasion) was assessed using a standard gentamicin survival assay, and epithelial release of IL-6 and IL-8 was measured using a bead based immunoassay. B. licheniformis was cytotoxic to NuLi-1 cells at 24 h. At 3 h post-challenge, B. licheniformis elicited high IL-6 and IL-8 inflammatory responses from NuLi-1 cells compared with cells treated with heat-treated geogenic dust PM10 (p < 0.0001). Whilst treatment of cells with B. licheniformis increased inflammation, this did not make the cells more susceptible to NTHi infection. This study highlights that geogenic dust PM10 can harbour viable bacterial spores that induce inflammation in respiratory epithelium. The impact on respiratory health from inhalation of bacterial spores in PM10 in arid environments may be underestimated. Further investigation into the contribution of B. licheniformis and the wider dust microbiome to respiratory infection is warranted.

Co-infection with Chikungunya virus alters trafficking of pathogenic CD8+ T cells into the brain and prevents Plasmodium-induced neuropathology.

Arboviral diseases have risen significantly over the last 40 years, increasing the risk of co-infection with other endemic disease such as malaria. However, nothing is known about the impact arboviruses have on the host response toward heterologous pathogens during co-infection. Here, we investigate the effects of Chikungunya virus (CHIKV) co-infection on the susceptibility and severity of malaria infection. Using the Plasmodium berghei ANKA (PbA) experimental cerebral malaria (ECM) model, we show that concurrent co-infection induced the most prominent changes in ECM manifestation. Concurrent co-infection protected mice from ECM mortality without affecting parasite development in the blood. This protection was mediated by the alteration of parasite-specific CD8+ T-cell trafficking through an IFNγ-mediated mechanism. Co-infection with CHIKV induced higher splenic IFNγ levels that lead to high local levels of CXCL9 and CXCL10. This induced retention of CXCR3-expressing pathogenic CD8+ T cells in the spleen and prevented their migration to the brain. This then averts all downstream pathogenic events such as parasite sequestration in the brain and disruption of blood-brain barrier that prevents ECM-induced mortality in co-infected mice.

Interferon regulatory factor 1 is essential for pathogenic CD8+ T cell migration and retention in the brain during experimental cerebral malaria.

Host immune response has a key role in controlling the progression of malaria infection. In the well-established murine model of experimental cerebral malaria (ECM) with Plasmodium berghei ANKA infection, proinflammatory Th1 and CD8+ T cell response are essential for disease development. Interferon regulatory factor 1 (IRF1) is a transcription factor that promotes Th1 responses, and its absence was previously shown to protect from ECM death. Yet the exact mechanism of protection remains unknown. Here we demonstrated that IRF1-deficient mice (IRF1 knockout) were protected from ECM death despite displaying early neurological signs. Resistance to ECM death was a result of reduced parasite sequestration and pathogenic CD8+ T cells in the brain. Further analysis revealed that IRF1 deficiency suppress interferon-γ production and delayed CD8+ T cell proliferation. CXCR3 expression was found to be decreased in pathogenic CD8+ T cells, which limited their migration to the brain. In addition, reduced expression of adhesion molecules by brain endothelial cells hampered leucocyte retention in the brain. Taken together, these factors limited sequestration of pathogenic CD8+ T cells and consequently its ability to induce extensive damage to the blood-brain barrier.

A Sensitive Method for Detecting Zika Virus Antigen in Patients' Whole-Blood Specimens as an Alternative Diagnostic Approach.

Background: Epidemics caused by the reemergence of Zika virus (ZIKV) warrant the need to develop new diagnostic measures to complement currently used detection methods. In this study, we explored the detection of ZIKV antigen in a defined leukocyte subset from patients' whole-blood specimens. Methods: Whole-blood samples were obtained at the acute and early convalescent phases from ZIKV-infected patients during the Singapore outbreak in August-September 2016. Presence of ZIKV antigen was determined by flow cytometry staining for intracellular ZIKV NS3, using a ZIKV-specific polyclonal antibody. The presence of ZIKV antigen was determined in CD45+CD14+ monocytes. Results: Data showed that ZIKV NS3 antigen could be detected in CD45+CD14+ monocytes. The levels of detection were further categorized into 3 groups: high (positivity among >40% of monocytes), moderate (positivity among 10%-40%), and low (positivity among <10%). While a majority of patients showed a decrease in the amount of ZIKV antigen detected at later time points, some patients displayed higher levels as the disease progressed. Conclusions: Our data highlights an alternative approach in using flow cytometry as a sensitive method for detecting ZIKV antigen in whole blood. Importantly, it further confirms the role of CD14+ monocytes as an important cellular target for ZIKV infection during the viremic phase.

Elevated levels of circulating exosome in COPD patients are associated with systemic inflammation.

Chronic obstructive pulmonary disease (COPD) is characterized by progressive pulmonary and systemic inflammation. Acute exacerbations of COPD (AECOPD) are associated with acute inflammation and infections and increase the rates of morbidity and mortality. Currently, neither the aetiology nor pathogenesis of AECOPD are entirely understood. Exosomes have been reported to regulate immunity and inflammation via specific intercellular communications through an array of macromolecules (e.g. microRNA and proteins) contained within these microvesicles. We evaluated the level of circulating exosomes in relation to systemic inflammation in patients with AECOPD (n = 20) or stable COPD (sCOPD; n = 20) in comparison to non-smoking healthy controls (n = 20). Exosomes in plasma were isolated by precipitation-based method, and quantified using a CD9 expression based enzyme-linked immunosorbent assay (ELISA). Plasma biomarkers of systemic inflammation, C-reactive protein (CRP), soluble tumour necrosis factor receptor-1 (sTNFR1) and interleukin (IL)-6 were also quantified using ELISA. Levels of plasma exosome were higher in AECOPD patients (p < 0.001) and sCOPD patients (p < 0.05) compared to controls. Plasma levels of CRP and sTNFR1 were highest in AECOPD, followed by sCOPD patients compared to healthy controls (p < 0.05). Plasma IL-6 was elevated in AECOPD (p < 0.05) and sCOPD patients (p < 0.01) compared to controls. The level of exosome correlated with the levels of CRP, sTNFR1 and IL-6 in plasma. Exosomes may therefore be involved in the inflammatory process of AECOPD. Further studies involving exosomal phenotyping and molecular characterization are required to fully understand their role in the pathophysiology of COPD.

Cross-reactive dengue human monoclonal antibody prevents severe pathologies and death from Zika virus infections.

Zika virus (ZIKV) infections have been linked with neurological complications and congenital Zika syndrome. Given the high level of homology between ZIKV and the related flavivirus dengue virus (DENV), we investigated the level of cross-reactivity with ZIKV using a panel of DENV human mAbs. A majority of the mAbs showed binding to ZIKV virions, with several exhibiting neutralizing capacities against ZIKV in vitro. Three of the best ZIKV-neutralizing mAbs were found to recognize diverse epitopes on the envelope (E) glycoprotein: the highly conserved fusion-loop peptide, a conformation-specific epitope on the E monomer, and a quaternary epitope on the virion surface. The most potent ZIKV-neutralizing mAb (SIgN-3C) was assessed in 2 type I interferon receptor-deficient (IFNAR-/-) mouse models of ZIKV infection. Treatment of adult nonpregnant mice with SIgN-3C rescued mice from virus-induced weight loss and mortality. The SIgN-3C variant with Leu-to-Ala mutations in the Fc region (SIgN-3C-LALA) did not induce antibody-dependent enhancement (ADE) in vitro but provided similar levels of protection in vivo. In pregnant ZIKV-infected IFNAR-/- mice, treatment with SIgN-3C or SIgN-3C-LALA significantly reduced viral load in the fetal organs and placenta and abrogated virus-induced fetal growth retardation. Therefore, SIgN-3C-LALA holds promise as a ZIKV prophylactic and therapeutic agent.

Fingolimod treatment abrogates chikungunya virus-induced arthralgia.

Chikungunya virus (CHIKV) is one of the many rheumatic arthropod-borne alphaviruses responsible for debilitating joint inflammation in humans. Despite the severity in many endemic regions, clinically approved intervention targeting the virus remains unavailable. CD4+ T cells have been shown to mediate CHIKV-induced joint inflammation in mice. We demonstrate here that transfer of splenic CD4+ T cells from virus-infected C57BL/6 mice into virus-infected T cell receptor-deficient (TCR-/-) mice recapitulated severe joint pathology including inflammation, vascular leakages, subcutaneous edema, and skeletal muscle necrosis. Proteome-wide screening identified dominant CD4+ T cell epitopes in nsP1 and E2 viral antigens. Transfer of nsP1- or E2-specific primary CD4+ T cell lines into CHIKV-infected TCR-/- recipients led to severe joint inflammation and vascular leakage. This pathogenic role of virus-specific CD4+ T cells in CHIKV infections led to the assessment of clinically approved T cell-suppressive drugs for disease intervention. Although drugs targeting interleukin-2 pathway were ineffective, treatment with fingolimod, an agonist of sphingosine 1-phosphate receptor, successfully abrogated joint pathology in CHIKV-infected animals by blocking the migration of CD4+ T cells into the joints without any effect on viral replication. These results set the stage for further clinical evaluation of fingolimod in the treatment of CHIKV-induced joint pathologies.