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Pneumonia is a severe complication caused by inflammation of the lungs following infection with seasonal and pandemic strains of influenza A virus (IAV) that can result in lung pathology, respiratory failure and death. There is currently no treatment available for severe disease and pneumonia caused by IAV. Antivirals are available, but they are far from satisfactory if treatment is not initiated within 48 hours of symptoms onset. Influenza complications and mortality are often associated with high viral load and excessive lung inflammatory cytokine response. Therefore, we simultaneously targeted IAV with the antiviral drug oseltamivir and inflammation with the anti-inflammatory drug etanercept, targeting TNF after the onset of clinical signs to treat IAV pneumonia effectively. The combined treatment effectively reduced lung viral load, lung pathology, morbidity and mortality during respiratory IAV infection in mice, contemporaneous with significant downregulation of the inflammatory cytokines TNF, IL-1β, IL-6, IL-12p40, chemokines CCL2, CCL5 and CXCL10 and dampened STAT3 activation. Consequently, combined therapy with oseltamivir and a STAT3 inhibitor also effectively reduced clinical disease and lung pathology. Combined treatment using either of the anti-inflammatory drugs and oseltamivir dampened an overlapping set of cytokines. Thus, combined therapy targeting a specific cytokine or cytokine signaling pathway plus an antiviral drug provides an effective treatment strategy for ameliorating IAV pneumonia. Effective treatment of IAV pneumonia required multiple doses of etanercept and a high dose of oseltamivir. This approach might apply to the treatment of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).Significance StatementAntivirals against influenza A virus (IAV) are ineffective in treating pneumonia if administered 48 h after onset of disease symptoms. The host inflammatory response and tissue damage caused by IAV are responsible for lung pathology. We reasoned that targeting both virus and inflammation would be more effective in reducing lung pathology and pneumonia, morbidity and mortality. The simultaneous treatment with an anti-inflammatory drug targeting TNF or STAT3, combined with the anti-IAV antiviral drug, oseltamivir, significantly improved clinical disease, reduced lung viral load and pathology, and protected mice from severe pneumonia. The combined treatment suppressed multiple pro-inflammatory cytokines and cytokine signaling pathways. Thus, after the onset of disease symptoms, both virus and inflammation must be targeted to treat IAV pneumonia effectively.

Viral causes of pneumonia pose constant threats to global public health, but there are no specific treatments currently available for the condition. Antivirals are ineffective when administered late after the onset of symptoms. Pneumonia is caused by an exaggerated inflammatory cytokine response to infection, but tissue necrosis and damage caused by virus also contribute to lung pathology. We hypothesized that viral pneumonia can be treated effectively if both virus and inflammation are simultaneously targeted. Combined treatment with the antiviral drug cidofovir and etanercept, which targets tumor necrosis factor (TNF), down-regulated nuclear factor kappa B-signaling and effectively reduced morbidity and mortality during respiratory ectromelia virus (ECTV) infection in mice even when treatment was initiated after onset of clinical signs. Treatment with cidofovir alone reduced viral load, but animals died from severe lung pathology. Treatment with etanercept had no effect on viral load but diminished levels of inflammatory cytokines and chemokines including TNF, IL-6, IL-1β, IL-12p40, TGF-β, and CCL5 and dampened activation of the STAT3 cytokine-signaling pathway, which transduces signals from multiple cytokines implicated in lung pathology. Consequently, combined treatment with a STAT3 inhibitor and cidofovir was effective in improving clinical disease and lung pathology in ECTV-infected mice. Thus, the simultaneous targeting of virus and a specific inflammatory cytokine or cytokine-signaling pathway is effective in the treatment of pneumonia. This approach might be applicable to pneumonia caused by emerging and re-emerging viruses, like seasonal and pandemic influenza A virus strains and severe acute respiratory syndrome coronavirus 2.

Influenza pneumonia is a severe complication caused by inflammation of the lungs following infection with seasonal and pandemic strains of influenza A virus (IAV), that can result in lung pathology, respiratory failure, and death. There is currently no treatment for severe disease and pneumonia caused by IAV. Antivirals are available but are only effective if treatment is initiated within 48 h of onset of symptoms. Influenza complications and mortality are often associated with high viral load and an excessive lung inflammatory cytokine response. Therefore, we simultaneously targeted the virus and inflammation. We used the antiviral oseltamivir and the anti-inflammatory drug etanercept to dampen TNF signaling after the onset of clinical signs to treat pneumonia in a mouse model of respiratory IAV infection. The combined treatment down-regulated the inflammatory cytokines TNF, IL-1β, IL-6, and IL-12p40, and the chemokines CCL2, CCL5, and CXCL10. Consequently, combined treatment with oseltamivir and a signal transducer and activator of transcription 3 (STAT3) inhibitor effectively reduced clinical disease and lung pathology. Combined treatment using etanercept or STAT3 inhibitor and oseltamivir dampened an overlapping set of cytokines. Thus, combined therapy targeting a specific cytokine or cytokine signaling pathway and an antiviral drug provide an effective treatment strategy for ameliorating IAV pneumonia. This approach might apply to treating pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Summary Multiple lines of evidence show that modern humans interbred with archaic Denisovans. Here, we report an account of shared demographic history between Australasians and Denisovans distinctively in Island Southeast Asia. Our analyses are based on ∼2.3 million genotypes from 118 ethnic groups of the Philippines, including 25 diverse self-identified Negrito populations, along with high-coverage genomes of Australopapuans and Ayta Magbukon Negritos. We show that Ayta Magbukon possess the highest level of Denisovan ancestry in the world—∼30%–40% greater than that of Australians and Papuans—consistent with an independent admixture event into Negritos from Denisovans. Together with the recently described Homo luzonensis, we suggest that there were multiple archaic species that inhabited the Philippines prior to the arrival of modern humans and that these archaic groups may have been genetically related. Altogether, our findings unveil a complex intertwined history of modern and archaic humans in the Asia-Pacific region, where distinct Islander Denisovan populations differentially admixed with incoming Australasians across multiple locations and at various points in time., Highlights • Comprehensive analyses of archaic ancestry among 118 Philippine ethnic groups • Ayta Magbukon display ∼30%–40% greater Denisovan ancestry than Australopapuans • The model is explained by a distinct admixture event into Negritos from Denisovans • Prior to modern humans, Islander Denisovans may have been present in the Philippines, Larena et al. reveal Philippine Ayta to possess the highest level of Denisovan ancestry in the world—∼30%–40% greater than that of Australopapuans—consistent with an independent admixture event into Negritos from Denisovans. The Philippine archipelago is thus likely inhabited by multiple archaic groups prior to the arrival of modern humans.

Significance A key link to understand human history in Island Southeast Asia is the Philippine archipelago and its poorly investigated genetic diversity. We analyzed the most comprehensive set of population-genomic data for the Philippines: 1,028 individuals covering 115 indigenous communities. We demonstrate that the Philippines were populated by at least five waves of human migration. The Cordillerans migrated into the Philippines prior to the arrival of rice agriculture, where some remain as the least admixed East Asians carrying an ancestry shared by all Austronesian-speaking populations, thereby challenging an exclusive out-of-Taiwan model of joint farming–language–people dispersal. Altogether, our findings portray the Philippines as a crucial gateway, with a multilayered history, that ultimately changed the genetic landscape of the Asia-Pacific region., Island Southeast Asia has recently produced several surprises regarding human history, but the region’s complex demography remains poorly understood. Here, we report ∼2.3 million genotypes from 1,028 individuals representing 115 indigenous Philippine populations and genome-sequence data from two ∼8,000-y-old individuals from Liangdao in the Taiwan Strait. We show that the Philippine islands were populated by at least five waves of human migration: initially by Northern and Southern Negritos (distantly related to Australian and Papuan groups), followed by Manobo, Sama, Papuan, and Cordilleran-related populations. The ancestors of Cordillerans diverged from indigenous peoples of Taiwan at least ∼8,000 y ago, prior to the arrival of paddy field rice agriculture in the Philippines ∼2,500 y ago, where some of their descendants remain to be the least admixed East Asian groups carrying an ancestry shared by all Austronesian-speaking populations. These observations contradict an exclusive “out-of-Taiwan” model of farming–language–people dispersal within the last four millennia for the Philippines and Island Southeast Asia. Sama-related ethnic groups of southwestern Philippines additionally experienced some minimal South Asian gene flow starting ∼1,000 y ago. Lastly, only a few lowlanders, accounting for

Ectromelia virus (ECTV) causes mousepox, a surrogate mouse model for smallpox caused by variola virus in humans. Both orthopoxviruses encode tumor necrosis factor receptor (TNFR) homologs or viral TNFR (vTNFR). These homologs are termed cytokine response modifier (Crm) proteins, containing a TNF-binding domain and a chemokine-binding domain called smallpox virus-encoded chemokine receptor (SECRET) domain. ECTV encodes one vTNFR known as CrmD. Infection of ECTV-resistant C57BL/6 mice with a CrmD deletion mutant virus resulted in uniform mortality due to excessive TNF secretion and dysregulated inflammatory cytokine production. CrmD dampened pathology, leukocyte recruitment, and inflammatory cytokine production in lungs including TNF, IL-6, IL-10, and IFN-γ. Blockade of TNF, IL-6, or IL-10R function with monoclonal antibodies reduced lung pathology and provided 60 to 100% protection from otherwise lethal infection. IFN-γ caused lung pathology only when both the TNF-binding and SECRET domains were absent. Presence of the SECRET domain alone induced significantly higher levels of IL-1β, IL-6, and IL-10, likely overcoming any protective effects that might have been afforded by anti–IFN-γ treatment. The use of TNF-deficient mice and those that express only membrane-associated but not secreted TNF revealed that CrmD is critically dependent on host TNF for its function. In vitro, recombinant Crm proteins from different orthopoxviruses bound to membrane-associated TNF and dampened inflammatory gene expression through reverse signaling. CrmD does not affect virus replication; however, it provides the host advantage by enabling survival. Host survival would facilitate virus spread, which would also provide an advantage to the virus., National Health and Medical Research Council of Australia to G.K. and G.C. (Grants 1007980 and 471426). The laboratory of A. Alcamí was funded by the Spanish Ministry of Science and Innovation and European Union (European Regional Development’s Funds)

Excessive tumor necrosis factor (TNF) is known to cause significant pathology. Paradoxically, deficiency in TNF (TNF(−/−)) also caused substantial pathology during respiratory ectromelia virus (ECTV) infection, a surrogate model for smallpox. TNF(−/−) mice succumbed to fulminant disease whereas wild-type mice, and those engineered to express only transmembrane TNF (mTNF), fully recovered. TNF deficiency did not affect viral load or leukocyte recruitment but caused severe lung pathology and excessive production of the cytokines interleukin (IL)-6, IL-10, transforming growth factor beta (TGF-β), and interferon gamma (IFN-γ). Short-term blockade of these cytokines significantly reduced lung pathology in TNF(−/−) mice concomitant with induction of protein inhibitor of activated STAT3 (PIAS3) and/or suppressor of cytokine signaling 3 (SOCS3), factors that inhibit STAT3 activation. Consequently, inhibition of STAT3 activation with an inhibitor reduced lung pathology. Long-term neutralization of IL-6 or TGF-β protected TNF(−/−) mice from an otherwise lethal infection. Thus, mTNF alone is necessary and sufficient to regulate lung inflammation but it has no direct antiviral activity against ECTV. The data indicate that targeting specific cytokines or cytokine-signaling pathways to reduce or ameliorate lung inflammation during respiratory viral infections is possible but that the timing and duration of the interventive measure are critical.