New mouse model to mimic immune response to Flu virus in elderly

This week Science published several research papers in immunology. One of these papers is the study from Yale School of Medicine showing that Tlr7^–/– × Mavs^–/– double KO mice expressing human anti-viral molecule Mx1 display susceptibility to influenza A virus in a caspase 1/11-dependent manner.

It is known that elderly individuals show susceptibility to influenza A virus. One reason for this susceptibility is a reduced ability of elderly immune system to produce type I IFNs in response to influenza A virus.

In contrast, aging mice do not show such susceptibility to influenza A virus. Mouse does not express Mx1, a dynamin-like guanosine triphosphatase that blocks primary transcription of influenza in humans. To make mouse model "usable" for  studying human response influenza A virus, the authors created Mx1⁺ mice. Mx1⁺ mice were resistant to experimental influenza A virus infection and this resistance was dependent on Tlr7 and Mavs (both molecules are involved in anti-viral response).

Interestingly, while Mx1⁺ mice double deficient for Tlr7^–/– × Mavs^–/– were susceptible to influenza A virus infection,  Mx1⁺ mice triple deficient for Tlr7^–/– × Mavs^–/–× Casp1/11^–/– were resistant to influenza A virus infection even though both showed similar viral burden (Of note, Tlr7^–/– × Mavs^–/–× Casp1/11^–/– mice were eventually cleared the virus by 30 days after infection).

Moreover, Mx1⁺ mice triple deficient for Tlr7^–/– × Mavs^–/–× Casp1/11^–/– were resistant to influenza A virus infection even though they too showed similar secondary bacterial "bloom" in their airways.

These data suggested that caspase 1/11 signaling reduced "tissue tolerance" [rather than increased anti-viral response] to influenza A virus infection and secondary bacterial "bloom" in Mx1⁺ mice deficient for anti-viral innate signaling via Tlr7^–/– × Mavs^–/–.

In summary, this study showed that intact caspase 1/11 signaling compromises tissue tolerance to acute influenza A virus infection and secondary bacterial "bloom" in host with a weakened anti-viral signaling.

David Usharauli

Complement C3's inside job

I was very excited when I saw first time a research article about novel function of complement C3. It was published in September 5 issue of journal Science and as the title suggested it showed as C3 having intracellular signaling capacity.

But after reading it, I am a little bit confused. Data presented in this paper do not necessarily support the authors conclusions, at least unequivocally. Surprisingly, the results in this paper are generated fully from the in vitro studies, that is by itself very rare, almost unheard of, for a Science paper with the focus on immunology. 

Moreover, if one considers an in vitro experiment, one expects to see an experimental design that incorporates specific gene knockout (KO) cells, minimum. However, not a single KO cells are employed. For specific gene inhibition or specific protein depletion, the authors have used either (a) siRNA or (b) chemical inhibitors or (3) serum depleted of specific factors or antibody. However, none of these treatments provide the "clean" results. 

Still, few results are worth mentioning, though with limitations discussed above.

First, using NF-kB-driven luciferase reporter HEK293T cell line, the authors showed that incubation of cells with adenovirus type 5 vector (AdV) in presence of serum (but not AdV alone) could activate NF-kB. Activation of NF-kB was equally diminished when serum was either (a) heat inactivated or (b) antibody depleted. However, activation of NF-kB was completely abolished when heat inactivated antibody depleted serum was used. The authors speculated that both antibodies and complement have unique functions in NF-kB activation when exposed to AdV.

Next, the authors showed that transfection of cells with beads incubated with C3fBfD (alternative complement activation), but not with beads or C3fBfD alone, could activate NF-kB.

Interestingly, using AdV-GFP construct, the authors showed that AdV+serum combination generated factors that inhibited AdV replication.

Mechanistically, using siRNA technology, the authors speculated that serum complement effect was mediated via MAVS (RNA recognizing intracellular molecule). However, the authors did not show whether C3 could interact directly with MAVS (for example, co-immunoprecipitation).

In summary, this paper tries to suggest that C3 attached to viruses or bacteria are internalized by the cells and signal through MAVS to activate NF-kB and produce cell autonomous defense  state.

David Usharauli

    

Reverse engineering the T-independent type 2 (TI-2) antibody responses

A very interesting and thought-provoking research in humoral immunity was published in journal Science this week. It came from Bruce Beutler's lab at the UT Southwestern Medical Center. Professor Beutler, as many may know, is a 2011 recipient of Nobel Prize in Physiology or Medicine for his discovery of endotoxin receptor (TLR4).

Actually, before I review this paper I would like to highlight that since 2005 when Ruslan Medzhitov's lab has published the first paper in Nature that suggested the role of TLR in B cell responses and then later in 2006, David Nemazee's lab has published new study in Science questioning the validity of 2005 paper, there was a controversy in this field. In fact, in 2009, Ruslan Medzhitov's lab has published another study that confirmed that haptenated proteins used in 2006 study could promote antibody response independent of TLR signaling. However the mechanism remained elusive.

It appears that this new study in Science may be a first glimpse in the darkness to reconcile the differences.

I also would like to point out that Professor Beutler's lab is famous for studying ENU-induced random mutations in mice to generate unbiased molecular signature of immune deficiencies.

Humoral immunity is divided into T-dependent and T-independent (TI) antibody response. TI responses, are in turn segregated into two categories: TI-1 and TI-2. This new study focus on TI-2 response.

Initially, using NP-Ficoll immunization, as a TI-2 model antigen, the authors observed that NP-specific IgM response was diminished in single STING-KO, cGAS-KO, MAVS-KO mice but not in several TLR signaling KO mice. 

Mice, double deficient in cGAS/MAVS, had almost complete absence of NP-specific IgM response.

This was strange observation since STING, cGAS and MAVS are known to detect the presence of DNAs or RNAs and NP-Ficoll contained none of them.

Using adoptive B cell transfer from these three KO mice into RAG-KO hosts, the authors showed that this effect of NP-Ficoll was B cell intrinsic (and the number of B1 or MZ B cells were normal in non-responding KOs).

The authors concluded that DNAs and RNAs were generated as a result of B cell stimulation by NP-Ficoll. Indeed, several endogenous retroviruses were up-regulated in NP+ B cells as compared to NP- B cells.

Furthermore, NP-ficoll immunization induced Reverse Transcriptase (RT) activity in NP+ B cells.

Parallel experiments revealed that mice deficient in NF-kB signaling also lacked the ability to respond to NP-Ficoll immunization or express endogenous retroviruses.

The authors observed that B cells from MAVS-KO, but not from cGAS KO mice, showed reduced phosphorylation of NF-kB proteins after anti-IgM stimulation, implying that MAVS played more critical role in sustained activation of NF-kB.

In summary, the authors proposed the new model of TI-2 antibody response. According to this model, when antigens with repetitive epitopes engage specific B cells, this activates initial wave of NF-kB activity leading to expression of endogenous retroviruses as a RNA that are subsequently converted into cDNA by RT activity. These newly generated RNAs and cDNAs are recognized by MAVS and cGAS/STING pathways, respectively. In turn, MAVS activation induces second wave of NF-kB activation to sustain TI-2 antibody response.

As the authors correctly pointed out, at this stage, it is not clear whether expression of endogenous retroviruses in activated B cells indeed play a critical role in TI-2 response. Their presence could be coincidental to this process. It would require B cell devoid of endogenous retroviruses to definitely test their precise role (since both TI-1 and T-dependent antibody response were shown to be STING, MAVS and cGAS independent).

For me this results represent new concept how we should assess the role of endogenous viruses present among our genes. Not everyone carries them, even among laboratory mice strains there is significant differences. Why would nature develop such system where TI-2 response would depend on endogenous viruses? It is just a fascinating idea.  

David Usharauli