Circadian rhythms of macrophages are altered by the acidic pH of the tumor microenvironment.

Macrophages are prime therapeutic targets due to their pro-tumorigenic and immunosuppressive functions in tumors, but varying efficacy of therapeutic approaches targeting macrophages highlights our incomplete understanding of how the tumor microenvironment (TME) can influence regulation of macrophages. The circadian clock is a key internal regulator of macrophage function, but how circadian rhythms of macrophages may be influenced by the tumor microenvironment remains unknown. We found that conditions associated with the TME such as polarizing stimuli, acidic pH, and elevated lactate concentrations can each alter circadian rhythms in macrophages. Circadian rhythms were enhanced in pro-resolution macrophages but suppressed in pro-inflammatory macrophages, while acidic pH had divergent effects on circadian rhythms depending on macrophage phenotype. While cyclic AMP (cAMP) has been reported to play a role in macrophage response to acidic pH, our results indicate that pH-driven changes in circadian rhythms are not mediated solely by the cAMP signaling pathway. Remarkably, clock correlation distance analysis of tumor-associated macrophages (TAMs) revealed evidence of circadian disorder in TAMs. This is the first report providing evidence that circadian rhythms of macrophages are altered within the TME. Our data suggest that heterogeneity in circadian rhythms at the population level may underlie this circadian disorder. Finally, we sought to determine how circadian regulation of macrophages impacts tumorigenesis, and found that tumor growth was suppressed when macrophages had a functional circadian clock. Our work demonstrates a novel mechanism by which the tumor microenvironment can influence macrophage biology through altering circadian rhythms, and the contribution of circadian rhythms in macrophages to suppressing tumor growth.

Do macrophages follow the beat of circadian rhythm in TIME (Tumor Immune Microenvironment)?

Advances in cancer research have made clear the critical role of the immune response in clearing tumors. This breakthrough in scientific understanding was heralded by the success of immune checkpoint blockade (ICB) therapies such as anti-programmed cell death protein 1 (PD-1)/ programmed death-ligand 1 (PD-L1) and anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), as well as the success of chimeric antigen receptor (CAR) T cells in treating liquid tumors. Thus, much effort has been made to further understand the role of the immune response in tumor progression, and how we may target it to treat cancer. Macrophages are a component of the tumor immune microenvironment (TIME) that can promote tumor growth both indirectly, by suppressing T cell responses necessary for tumor killing, as well as directly, through deposition of extracellular matrix and promotion of angiogenesis. Thus, understanding regulation of macrophages within the tumor microenvironment (TME) is key to targeting them for immunotherapy. However, circadian rhythms (24-hour cycles) are a fundamental aspect of macrophage biology that have yet to be investigated for their role in macrophage-mediated suppression of the anti-tumor immune response Circadian rhythms regulate macrophage-mediated immune responses through time-of-day-dependent regulation of macrophage function. A better understanding of the circadian biology of macrophages in the context of the TME may allow us to exploit synergy between existing and upcoming treatments and circadian regulation of immunity.

MYC Ran Up the Clock: The Complex Interplay between MYC and the Molecular Circadian Clock in Cancer.

The MYC oncoprotein and its family members N-MYC and L-MYC are known to drive a wide variety of human cancers. Emerging evidence suggests that MYC has a bi-directional relationship with the molecular clock in cancer. The molecular clock is responsible for circadian (~24 h) rhythms in most eukaryotic cells and organisms, as a mechanism to adapt to light/dark cycles. Disruption of human circadian rhythms, such as through shift work, may serve as a risk factor for cancer, but connections with oncogenic drivers such as MYC were previously not well understood. In this review, we examine recent evidence that MYC in cancer cells can disrupt the molecular clock; and conversely, that molecular clock disruption in cancer can deregulate and elevate MYC. Since MYC and the molecular clock control many of the same processes, we then consider competition between MYC and the molecular clock in several select aspects of tumor biology, including chromatin state, global transcriptional profile, metabolic rewiring, and immune infiltrate in the tumor. Finally, we discuss how the molecular clock can be monitored or diagnosed in human tumors, and how MYC inhibition could potentially restore molecular clock function. Further study of the relationship between the molecular clock and MYC in cancer may reveal previously unsuspected vulnerabilities which could lead to new treatment strategies.

Publisher Correction: Natural and directed antigenic drift of the H1 influenza virus hemagglutinin stalk domain.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Publisher Correction: Natural and directed antigenic drift of the H1 influenza virus hemagglutinin stalk domain.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Natural and directed antigenic drift of the H1 influenza virus hemagglutinin stalk domain.

The induction of antibodies specific for the influenza HA protein stalk domain is being pursued as a universal strategy against influenza virus infections. However, little work has been done looking at natural or induced antigenic variability in this domain and the effects on viral fitness. We analyzed human H1 HA head and stalk domain sequences and found substantial variability in both, although variability was highest in the head region. Furthermore, using human immune sera from pandemic A/California/04/2009 immune subjects and mAbs specific for the stalk domain, viruses were selected in vitro containing mutations in both domains that partially contributed to immune evasion. Recombinant viruses encoding amino acid changes in the HA stalk domain replicated well in vitro, and viruses incorporating two of the stalk mutations retained pathogenicity in vivo. These findings demonstrate that the HA protein stalk domain can undergo limited drift under immune pressure and the viruses can retain fitness and virulence in vivo, findings which are important to consider in the context of vaccination targeting this domain.

Antigenicity of the 2015-2016 seasonal H1N1 human influenza virus HA and NA proteins.

Antigenic drift of the hemagglutinin (HA) and neuraminidase (NA) influenza virus proteins contributes to reduced vaccine efficacy. To analyze antigenic drift in human seasonal H1N1 viruses derived from the 2009 pandemic H1N1 virus (pH1N1-like viruses) accounts for the limited effectiveness (around 40%) of vaccination against pH1N1-like viruses during the 2015-2016 season, nasal washes/swabs collected from adult subjects in the Rochester, NY area, were used to sequence and isolate the circulating viruses. The HA and NA proteins from viruses circulating during the 2015-2016 season encoded eighteen and fourteen amino acid differences, respectively, when compared to A/California/04/2009, a strain circulating at the origin of the 2009 pandemic. The circulating strains belonged to subclade 6B.1, defined by HA amino acid substitutions S101N, S179N, and I233T. Hemagglutination-inhibiting (HAI) and HA-specific neutralizing serum antibody (Ab) titers from around 50% of pH1N1-like virus-infected subjects and immune ferrets were 2-4 fold lower for the 2015-2016 circulating strains compared to the vaccine strain. In addition, using a luminex-based mPlex HA assay, the binding of human sera from subjects infected with pH1N1-like viruses to the HA proteins from circulating and vaccine strains was not identical, strongly suggesting antigenic differences in the HA protein. Additionally, NA inhibition (NAI) Ab titers in human sera from pH1N1-like virus-infected subjects increased after the infection and there were measurable antigenic differences between the NA protein of circulating strains and the vaccine strain using both ferret and human antisera. Despite having been vaccinated, infected subjects exhibited low HAI Ab titers against the vaccine and circulating strains. This suggests that poor responses to the H1N1 component of the vaccine as well as antigenic differences in the HA and NA proteins of currently circulating pH1N1-like viruses could be contributing to risk of infection even after vaccination.

Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses.

In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of the pH1N1 NS1 protein to naturally acquire mutations that restore this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and proinflammatory responses to a greater extent than that with the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. The pH1N1/NSs-6mut virus grew similarly to pH1N1/NSs-wt in mouse lungs, but infection with pH1N1/NSs-6mut induced lower levels of proinflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower level of inflammation induced by the pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis.IMPORTANCE Seasonal influenza A viruses (IAVs) are among the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when IAVs circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people then and up to the present. It was previously shown that the NS1 protein from the 2009 pandemic H1N1 (pH1N1) virus is not able to inhibit general gene expression. However, currently circulating pH1N1 viruses have evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) that allow the NS1 protein of contemporary pH1N1 strains to inhibit host gene expression, which correlates with its ability to interact with CPSF30. Infection with a recombinant pH1N1 virus encoding these 6 amino acid changes (pH1N1/NSs-6mut) induced lower levels of proinflammatory cytokines, resulting in viral attenuation in vivo This might represent an adaptation of pH1N1 virus to humans.